Polynucleotides encoding signal peptide-containing molecules

ABSTRACT

The invention provides human signal peptide-containing proteins (HSPP) and polynucleotides which identify and encode HSPP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of HSPP.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.11/905,820, filed Oct. 4, 2007, which is a divisional application ofU.S. patent application Ser. No. 10/820,474, filed Apr. 7, 2004, whichis a divisional of U.S. patent application Ser. No. 09/720,533, filedMar. 20, 2001, which is the National Phase of International ApplicationNo. PCT/US99/14484, filed Jun. 25, 1999, which claims benefit under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/090,762,filed Jun. 26, 1998, U.S. Provisional Patent Application No. 60/094,983,filed Jul. 31, 1998, U.S. Provisional Patent Application No. 60/102,686,filed Oct. 1, 1998, and U.S. Provisional Patent Application No.60/112,129, filed Dec. 11, 1998. These applications are all incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences of humansignal peptide-containing proteins and to the use of these sequences inthe diagnosis, treatment, and prevention of cell proliferative disordersincluding cancer; inflammation; and cardiovascular, neurological,reproductive, and developmental disorders.

BACKGROUND OF THE INVENTION

Protein transport is essential for cellular function. Transport of aprotein may be mediated by a signal peptide located at the aminoterminus of the protein itself. The signal peptide is comprised of aboutten to twenty hydrophobic amino acids which target the nascent proteinfrom the ribosome to a particular membrane bound compartment such as theendoplasmic reticulum (ER). Proteins targeted to the ER may eitherproceed through the secretory pathway or remain in any of the secretoryorganelles such as the ER, Golgi apparatus, or lysosomes. Proteins thattransit through the secretory pathway are either secreted into theextracellular space or retained in the plasma membrane. Secretedproteins are often synthesized as inactive precursors that are activatedby post-translational processing events during transit through thesecretory pathway. Such events include glycosylation, phosphorylation,proteolysis, and removal of the signal peptide by a signal peptidase.Other events that may occur during protein transport includechaperone-dependent unfolding and folding of the nascent protein andinteraction of the protein with a receptor or pore complex. Examples ofsecreted proteins with amino terminal signal peptides are discussedbelow and include receptors, extracellular matrix molecules, cytokines,hormones, growth and differentiation factors, neuropeptides,vasomediators, phosphokinases, phosphatases, phospholipases,phosphodiesterases, G and Ras-related proteins, ion channels,transporters/pumps, proteases, and transcription factors. (Reviewed inAlberts, B. et al. (1994) Molecular Biology of The Cell, GarlandPublishing, New York, N.Y., pp. 557-560, 582-592.)

G-protein coupled receptors (GPCRs) comprise a superfamily of integralmembrane proteins which transduce extracellular signals. GPCRs includereceptors for biogenic amines such as dopamine, epinephrine, histamine,glutamate (metabotropic effect), acetylcholine (muscarinic effect), andserotonin; for lipid mediators of inflammation such as prostaglandins,platelet activating factor, and leukotrienes; for peptide hormones suchas calcitonin, C5a anaphylatoxin, follicle stimulating hormone,gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin; andfor sensory signal mediators such as retinal photopigments and olfactorystimulatory molecules. The structure of these highly conserved receptorsconsists of seven hydrophobic transmembrane regions, cysteine disulfidebridges between the second and third extracellular loops, anextracellular N-terminus, and a cytoplasmic C-terminus. The N-terminusinteracts with ligands, the disulfide bridges interact with agonists andantagonists, and the large third intracellular loop interacts with Gproteins to activate second messengers such as cyclic AMP, phospholipaseC, inositol triphosphate, or ion channels. (Reviewed in Watson, S, andArkinstall, S. (1994) The G-protein Linked Receptor Facts Book, AcademicPress, San Diego, Calif., pp. 2-6; and Bolander, F. F. (1994) MolecularEndocrinology, Academic Press, San Diego, Calif., pp. 162-176.)

Other types of receptors include cell surface antigens identified onleukocytic cells of the immune system. These antigens have beenidentified using systematic, monoclonal antibody (mAb)-based “shot gun”techniques. These techniques have resulted in the production of hundredsof mAbs directed against unknown cell surface leukocytic antigens. Theseantigens have been grouped into “clusters of differentiation” based oncommon immunocytochemical localization patterns in variousdifferentiated and undifferentiated leukocytic cell types. Antigens in agiven cluster are presumed to identify a single cell surface protein andare assigned a “CD” number. Some of the genes encoding proteinsidentified by CD antigens have been isolated and characterized as bothtransmembrane proteins and cell surface proteins anchored to the plasmamembrane via covalent attachment to fatty acid-containing glycolipidssuch as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A. N.et al. (1993) The Leucocyte Antigen Facts Book, Academic Press, SanDiego, Calif., pp. 144-145; Noel, L. S. et al. (1998) J. Biol. Chem.273:3878-3883.)

Tetraspanins are a superfamily of membrane proteins which facilitate theformation and stability of cell-surface signaling complexes containinglineage-specific proteins, integrins, and other tetraspanins. They areinvolved in cell activation, proliferation (including cancer),differentiation, adhesion, and motility. These proteins cross themembrane four times, have conserved intracellular—and C-termini and anextracellular, non-conserved hydrophilic domain. Tetraspanins include,e.g., platelet and endothelial cell membrane proteins, leukocyte surfaceproteins, tissue specific and tumorous antigens, and the retinitispigmentosa-associated gene peripherin. (Maecker, H. T. et al. (1997)FASEB J. 11:428-442.)

Matrix proteins (MPs) are transmembrane and extracellular proteins whichfunction in formation, growth, remodeling, and maintenance of tissuesand as important mediators and regulators of the inflammatory response.The expression and balance of MPs may be perturbed by biochemicalchanges that result from congenital, epigenetic, or infectious diseases.In addition, MPs affect leukocyte migration, proliferation,differentiation, and activation in the immune response. MPs arefrequently characterized by the presence of one or more domains whichmay include collagen-like domains, EGF-like domains, immunoglobulin-likedomains, and fibronectin-like domains. In addition, some MPs are heavilyglycosylated. MPs include extracellular proteins such as fibronectin,collagen, and galectin and cell adhesion receptors such as cell adhesionmolecules (CAMs), cadherins, and integrins. (Reviewed in Ayad, S. et al.(1994) The Extracellular Matrix Facts Book, Academic Press, San Diego,Calif., pp. 2-16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417;Sjaastad, M. D. and Nelson, W. J. (1997) BioEssays 19:47-55.)

Lectins are proteins characterized by their ability to bindcarbohydrates on cell membranes by means of discrete, modularcarbohydrate recognition domains, CRDs. (Kishore, U. et al. (1997)Matrix Biol. 15:583-592.) Certain cytokines and membrane-spanningproteins have CRDs which may enhance interactions with extracellular orintracellular ligands, with proteins in secretory pathways, or withmolecules in signal transduction pathways. The lipocalin superfamilyconstitutes a phylogenetically conserved group of more than fortyproteins that function by binding to and transporting a variety ofphysiologically important ligands. (Tanaka, T. et al. (1997) J. Biol.Chem. 272:15789-15795; and van't H of, W. et al. (1997) J. Biol. Chem.272:1837-1841.) Selectins are a family of calcium ion-dependent lectinsexpressed on inflamed vascular endothelium and the surface of someleukocytes. (Rossiter, H. et al. (1997) Mol. Med. Today 3:214-222.)

Protein kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Reversible protein phosphorylation is a key strategy forcontrolling protein functional activity in eukaryotic cells. The highenergy phosphate which drives this activation is generally transferredfrom adenosine triphosphate molecules (ATP) to a particular protein byprotein kinases and removed from that protein by protein phosphatases.Phosphorylation occurs in response to extracellular signals, cell cyclecheckpoints, and environmental or nutritional stresses. Protein kinasesmay be roughly divided into two groups; protein tyrosine kinases (PTKs)which phosphorylate tyrosine residues, and serine/threonine kinases(STKs) which phosphorylate serine or threonine residues. A few proteinkinases have dual specificity. A majority of kinases contain a similar250-300 amino acid catalytic domain. (Hardie, G. and Hanks, S. (1995)The Protein Kinase Facts Book, Vol I, pp. 7-47, Academic Press, SanDiego, Calif.)

Protein phosphatases remove phosphate groups from molecules previouslymodified by protein kinases thus participating in cell signaling,proliferation, differentiation, contacts, and oncogenesis. Proteinphosphorylation is a key strategy used to control protein functionalactivity in eukaryotic cells. The high energy phosphate is transferredfrom ATP to a protein by protein kinases and removed by proteinphosphatases. There appear to be three, evolutionarily-distinct proteinphosphatase gene families: protein phosphatases (PPs); protein tyrosinephosphatases (PTPs); and acid/alkaline phosphatases (APs). PPsdephosphorylate phosphoserine/threonine residues and are an importantregulator of many cAMP mediated, hormone responses in cells. PTPsreverse the effects of protein tyrosine kinases and therefore play asignificant role in cell cycle and cell signaling processes. AlthoughAPs dephosphorylate substrates in vitro, their role in vivo is not wellknown. (Charbonneau, H. and Tonks, N. K. (1992) Annu. Rev. Cell Biol.8:463-493.)

Cyclic nucleotides (cAMP and cGMP) function as intracellular secondmessengers to transduce a variety of extracellular signals, includinghormones, light and neurotransmitters. Cyclic nucleotidephosphodiesterases (PDEs) degrade cyclic nucleotides to theircorresponding monophosphates, thereby regulating the intracellularconcentrations of cyclic nucleotides and their effects on signaltransduction. At least seven families of mammalian PDEs have beenidentified based on substrate specificity and affinity, sensitivity tocofactors and sensitivity to inhibitory drugs. (Beavo, J. A. (1995)Physiological Reviews 75: 725-748.)

Phospholipases (PLs) are enzymes that catalyze the removal of fatty acidresidues from phosphoglycerides. PLs play an important role intransmembrane signal transduction and are named according to thespecific ester bond in phosphoglycerides that is hydrolyzed, i.e., A₁,A₂, C or D. PLA₂ cleaves the ester bond at position 2 of the glycerolmoiety of membrane phospholipids giving rise to arachidonic acid.Arachidonic acid is the common precursor to four major classes ofeicosanoids, namely prostaglandins, prostacyclins, thromboxanes andleukotrienes. Eicosanoids are signaling molecules involved in thecontraction of smooth muscle, platelet aggregation, and pain andinflammatory responses. (Alberts, B. et al. (1994) Molecular Biology ofThe Cell, Garland Publishing, Inc., New York, N.Y., pp. 85, 211,239-240, 642-645.)

The nucleotide cyclases, i.e., adenylate and guanylate cyclase, catalyzethe synthesis of the cyclic nucleotides, cAMP and cGMP, from ATP andGTP, respectively. They act in concert with phosphodiesterases, whichdegrade cAMP and cGMP, to regulate the cellular levels of thesemolecules and their functions. cAMP and cGMP function as intracellularsecond messengers to transduce a variety of extracellular signals, e.g.,hormones, and light and neurotransmitters. (Stryer, L. (1988)Biochemistry W. H. Freeman and Co., New York, pp. 975-980, 1029-1035.)

Cytokines are produced in response to cell perturbation. Some cytokinesare produced as precursor forms, and some form multimers in order tobecome active. They are produced in groups and in patternscharacteristic of the particular stimulus or disease, and the members ofthe group interact with one another and other molecules to produce anoverall biological response. Interleukins, neurotrophins, growthfactors, interferons, and chemokines are all families of cytokines whichwork in conjunction with cellular receptors to regulate cellproliferation and differentiation and to affect such activities asleukocyte migration and function, hematopoietic cell proliferation,temperature regulation, acute response to infections, tissue remodeling,apoptosis, and cell survival. Studies using antibodies or other drugsthat modify the activity of a particular cytokine are used to elucidatethe roles of individual cytokines in pathology and physiology.

Chemokines, in particular, are small chemoattractant cytokines involvedin inflammation, leukocyte proliferation and migration, angiogenesis andangiostasis, regulation of hematopoiesis, HIV infectivity, andstimulation of cytokine secretion. Chemokines generally contain 70-100amino acids and are subdivided into four subfamilies based on thepresence of conserved cysteine-based motifs. (Callard, R. and Gearing,A. (1994) The Cytokine Facts Book, Academic Press, New York, N.Y., pp.181-190, 210-213, 223-227.)

Growth and differentiation factors are secreted proteins which functionin intercellular communication. Some factors require oligomerization orassociation with MPs for activity. Complex interactions among thesefactors and their receptors trigger intracellular signal transductionpathways that stimulate or inhibit cell division, cell differentiation,cell signaling, and cell motility. Most growth and differentiationfactors act on cells in their local environment (paracrine signaling).There are three broad classes of growth and differentiation factors. Thefirst class includes the large polypeptide growth factors such asepidermal growth factor, fibroblast growth factor, transforming growthfactor, insulin-like growth factor, and platelet-derived growth factor.The second class includes the hematopoietic growth factors such as thecolony stimulating factors (CSFs). Hematopoietic growth factorsstimulate the proliferation and differentiation of blood cells such asB-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils,basophils, neutrophils, macrophages, and their stem cell precursors. Thethird class includes small peptide factors such as bombesin,vasopressin, oxytocin, endothelin, transferrin, angiotensin II,vasoactive intestinal peptide, and bradykinin which function as hormonesto regulate cellular functions other than proliferation.

Growth and differentiation factors play critical roles in neoplastictransformation of cells in vitro and in tumor progression in vivo.Inappropriate expression of growth factors by tumor cells may contributeto vascularization and metastasis of melanotic tumors. Duringhematopoiesis, growth factor misregulation can result in anemias,leukemias, and lymphomas. Certain growth factors such as interferon arecytotoxic to tumor cells both in vivo and in vitro. Moreover, somegrowth factors and growth factor receptors are related both structurallyand functionally to oncoproteins. In addition, growth factors affecttranscriptional regulation of both proto-oncogenes and oncosuppressorgenes. (Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRCPress, Ann Arbor, Mich., pp. 1-9.)

Proteolytic enzymes or proteases either activate or deactivate proteinsby hydrolyzing peptide bonds. Proteases are found in the cytosol, inmembrane-bound compartments, and in the extracellular space. The majorfamilies are the zinc, serine, cysteine, thiol, and carboxyl proteases.

Zinc proteases, e.g., carboxypeptidase A, have a zinc ion bound to theactive site. These proteases recognize C-terminal residues that containan aromatic or bulky aliphatic side chain, and hydrolyze the peptidebond adjacent to the C-terminal residues. Serine proteases have anactive site serine residue and include digestive enzymes, e.g., trypsinand chymotrypsin, components of the complement and blood-clottingcascades, and enzymes that control the degradation and turnover ofextracellular matrix (ECM) molecules. Cysteine proteases (e.g.cathepsin) are produced by monocytes, macrophages and other immunecells, and are involved in diverse cellular processes ranging from theprocessing of precursor proteins to intracellular degradation.Overproduction of these enzymes can cause the tissue destructionassociated with rheumatoid arthritis and asthma. Thiol proteases, e.g.,papain, contain an active site cysteine and are widely distributedwithin tissues. Carboxyl proteases, e.g., pepsin, are active only underacidic conditions (pH 2 to 3).

Guanosine triphosphate-binding proteins (G proteins) can be grouped intotwo major classes: heterotrimeric G proteins and small G proteins.Heterotrimeric G proteins interact with GPCRs that respond to hormones,growth factors, neuromodulators, or other signaling molecules. Theinteraction between GPCR and G protein allows the G protein to exchangeGTP for guanosine diphosphate (GDP). This exchange activates the Gprotein, allowing it to dissociate from the receptor and interact withthe its cognate second messenger-generating protein, e.g., adenylatecyclase, guanylate cyclase, phospholipase C, or ion channels. Thehydrolysis of GTP to GDP by the G protein acts as an on-off switch,terminating the action of the G protein and preparing it to interactwith another receptor molecule, thus beginning another round of signaltransduction.

The small G proteins consist of single 21-30 kDa polypeptides. They canbe classified into five subfamilies: Ras, Rho, Ran, Rab, andADP-ribosylation factor. These proteins regulate cell growth, cell cyclecontrol, protein secretion, and intracellular vesicle interaction. Inparticular, the Ras proteins are essential in transducing signals fromreceptor tyrosine kinases to serine/threonine kinases which control cellgrowth and differentiation. Mutant Ras proteins, which bind but can nothydrolyze GTP, are permanently activated and cause continuous cellproliferation or cancer. All five subfamilies share common structuralfeatures and four conserved motifs. Most of the membrane-bound Gproteins require a carboxy terminal isoprenyl group (CAAX), addedposttranslationally, for membrane association and biological activity.The G proteins also have a variable effector region, located betweenmotifs I and II, which is characterized as the interaction site forguanine nucleotide exchange factors or GTPase-activating proteins.

Eukaryotic cells are bound by a membrane and subdivided intomembrane-bound compartments. Membranes are impermeable to many ions andpolar molecules, therefore transport of these molecules is mediated byion channels, ion pumps, transport proteins, or pumps. Symporters andantiporters regulate cytosolic pH by transporting ions and smallmolecules, e.g., amino acids, glucose, and drugs, across membranes;symporters transport small molecules and ions in the same direction, andantiporters, in the opposite direction. Transporter superfamiliesinclude facilitative transporters and active ATP binding cassettetransporters involved in multiple-drug resistance and the targeting ofantigenic peptides to MHC Class I molecules. These transporters bind toa specific ion or other molecule and undergo conformational changes inorder to transfer the ion or molecule across a membrane. Transport canoccur by a passive, concentration-dependent mechanism or can be linkedto an energy source such as ATP hydrolysis or an ion gradient.

Ion channels, ion pumps, and transport proteins mediate the transport ofmolecules across cellular membranes. Symporters and antiporters regulatecytosolic pH by transporting ions and small molecules such as aminoacids, glucose, and drugs. Symporters transport small molecules and ionsunidirectionally, and antiporters, bidirectionally. Transportersuperfamilies include facilitative transporters and active ATP-bindingcassette transporters which are involved in multiple-drug resistance andthe targeting of antigenic peptides to MHC Class I molecules. Thesetransporters bind to a specific ion or other molecule and undergo aconformational change in order to transfer the ion or molecule acrossthe membrane. Transport can occur by a passive, concentration-dependentmechanism or can be linked to an energy source such as ATP hydrolysis.(Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell,Garland Publishing, New York, N.Y., pp. 523-546.)

Ion channels are formed by transmembrane proteins which create a linedpassageway across the membrane through which water and ions, such asNa⁺, K⁺, Ca²⁺, and Cl—, enter and exit the cell. For example, chloridechannels are involved in the regulation of the membrane electricpotential as well as absorption and secretion of ions across themembrane. Chloride channels also regulate the internal pH ofmembrane-bound organelles.

Ion pumps are ATPases which actively maintain membrane gradients. Ionpumps are classified as P, V, or F according to their structure andfunction. All have one or more binding sites for ATP in their cytosolicdomains. The P-class ion pumps include Ca²⁺ ATPase and Na⁺/K⁺ ATPase andfunction in transporting H⁺, Na⁺, K⁺, and Ca²⁺ ions. P-class pumpsconsist of two ÿ and two ÿ transmembrane subunits. The V- and F-classion pumps have similar structures and but transport only H⁺. F class H⁺pumps mediate transport across the membranes of mitochondria andchloroplasts, while V-class H⁺ pumps regulate acidity inside lysosomes,endosomes, and plant vacuoles.

A family of structurally related intrinsic membrane proteins known asfacilitative glucose transporters catalyze the movement of glucose andother selected sugars across the plasma membrane. The proteins in thisfamily contain a highly conserved, large transmembrane domain comprisedof 12 ÿ-helices, and several weakly conserved, cytoplasmic andexoplasmic domains (Pessin, J. E., and Bell, G. I. (1992) Annu. Rev.Physiol. 54:911-930).

Amino acid transport is mediated by Na⁺ dependent amino acidtransporters. These transporters are involved in gastrointestinal andrenal uptake of dietary and cellular amino acids and in neuronalreuptake of neurotransmitters. Transport of cationic amino acids ismediated by the system y+ family and the cationic amino acid transporter(CAT) family. Members of the CAT family share a high degree of sequencehomology, and each contains 12-14 putative transmembrane domains (Ito,K. and Groudine, M. (1997) J. Biol. Chem. 272:26780-26786).

Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1and PEPT 2 are responsible for gastrointestinal absorption and for renalreabsorbtion of peptides using an electrochemical H⁺ gradient as thedriving force. A heterodimeric peptide transporter, consisting of TAP 1and TAP 2, is associated with antigen processing. Peptide antigens aretransported across the membrane of the endoplasmic reticulum so they canbe presented to the major histocompatibility complex class I molecules.Each TAP protein consists of multiple hydrophobic membrane spanningsegments and a highly conserved ATP-binding cassette. (Boll, M. et al.(1996) Proc. Natl. Acad. Sci. 93:284-289.)

Hormones are secreted molecules that travel through the circulation andbind to specific receptors on the surface of, or within, target cells.Although they have diverse biochemical compositions and mechanisms ofaction, hormones can be grouped into two categories. One categoryconsists of small lipophilic hormones that diffuse through the plasmamembrane of target cells, bind to cytosolic or nuclear receptors, andform a complex that alters gene expression. Examples of these moleculesinclude retinoic acid, thyroxine, and the cholesterol-derived steroidhormones such as progesterone, estrogen, testosterone, cortisol, andaldosterone. The second category consists of hydrophilic hormones thatfunction by binding to cell surface receptors that transduce signalsacross the plasma membrane. Examples of such hormones include amino acidderivatives such as catecholamines and peptide hormones such asglucagon, insulin, gastrin, secretin, cholecystokinin,adrenocorticotropic hormone, follicle stimulating hormone, luteinizinghormone, thyroid stimulating hormone, and vasopressin. (See, forexample, Lodish et al. (1995) Molecular Cell Biology, ScientificAmerican Books Inc., New York, N.Y., pp. 856-864.)

Neuropeptides and vasomediators (NP/VM) comprise a large family ofendogenous signaling molecules. Included in this family areneuropeptides and neuropeptide hormones such as bombesin, neuropeptideY, neurotensin, neuromedin N, melanocortins, opioids, galanin,somatostatin, tachykinins, urotensin II and related peptides involved insmooth muscle stimulation, vasopressin, vasoactive intestinal peptide,and circulatory system-borne signaling molecules such as angiotensin,complement, calcitonin, endothelins, formyl-methionyl peptides,glucagon, cholecystokinin and gastrin. NP/VMs can transduce signalsdirectly, modulate the activity or release of other neurotransmittersand hormones, and act as catalytic enzymes in cascades. The effects ofNP/VMs range from extremely brief to long-lasting. (Reviewed in Martin,C. R. et al. (1985) Endocrine Physiology, Oxford University Press, NewYork, N.Y., pp. 57-62.)

Regulatory molecules turn individual genes or groups of genes on and offin response to various inductive mechanisms of the cell or organism; actas transcription factors by determining whether or not transcription isinitiated, enhanced, or repressed; and splice transcripts as dictated ina particular cell or tissue. Although they interact with short stretchesof DNA scattered throughout the entire genome, most gene expression isregulated near the site at which transcription starts or within the openreading frame of the gene being expressed. Many of the transcriptionfactors incorporate one of a set of DNA-binding structural motifs, eachof which contains either ÿ helices or β sheets and binds to the majorgroove of DNA. (Pabo, C. O. and R. T. Sauer (1992) Ann. Rev. Biochem.61:1053-95.) Other domains of transcription factors may form crucialcontacts with the DNA. In addition, accessory proteins provide importantinteractions which may convert a particular protein complex to anactivator or a repressor or may prevent binding. (Alberts, B. et al.(1994) Molecular Biology of the Cell, Garland Publishing Co, New York,N.Y. pp. 401-474.)

The discovery of new human signal peptide-containing proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of cell proliferative disorders including cancer;inflammation; and cardiovascular, neurological, reproductive, anddevelopmental disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, proteinswith signal peptides, referred to collectively as “HSPP” andindividually as “HSPP-1”, “HSPP-2”, “HSPP-3”, “HSPP-4”, “HSPP-5”,“HSPP-6”, “HSPP-7”, “HSPP-8”, “HSPP-9”, “HSPP-10”, “HSPP-11”, “HSPP-12”,“HSPP-13”, “HSPP-14”, “HSPP-15”, “HSPP-16”, “HSPP-17”, “HSPP-18”,“HSPP-19”, “HSPP-20”, “HSPP-21”, “HSPP-22”, “HSPP-23”, “HSPP-24”,“HSPP-25”, “HSPP-26”, “HSPP-27”, “HSPP-28”, “HSPP-29”, “HSPP-30”,“HSPP-31”, “HSPP-32”, “HSPP-33”, “HSPP-34”, “HSPP-35”, “HSPP-36”,“HSPP-37”, “HSPP-38”, “HSPP-39”, “HSPP-40”, “HSPP-41”, “HSPP-42”,“HSPP-43”, “HSPP-44”, “HSPP-45”, “HSPP-46”, “HSPP-47”, “HSPP-48”,“HSPP-49”, “HSPP-50”, “HSPP-51”, “HSPP-52”, “HSPP-53”, “HSPP-54”,“HSPP-55”, “HSPP-56”, “HSPP-57”, “HSPP-58”, “HSPP-59”, “HSPP-60”,“HSPP-61”, “HSPP-62”, “HSPP-63”, “HSPP-64”, “HSPP-65”, “HSPP-66”,“HSPP-67”, “HSPP-68”, “HSPP-69”, “HSPP-70”, “HSPP-71”, “HSPP-72”,“HSPP-73”, “HSPP-74”, “HSPP-75”, HSPP-76″, “HSPP-77”, “HSPP-78”,“HSPP-79”, “HSPP-80”, “HSPP-81”, “HSPP-82”, “HSPP-83”, “HSPP-84”,“HSPP-85”, “HSPP-86”, “HSPP-87”, “HSPP-88”, “HSPP-89”, “HSPP-90”,“HSPP-91”, “HSPP-92”, “HSPP-93”, “HSPP-94”, “HSPP-95”, “HSPP-96”,“HSPP-97”, “HSPP-98”, “HSPP-99”, “HSPP-100”, “HSPP-101”, “HSPP-102”,“HSPP-103”, “HSPP-104”, “HSPP-105”, “HSPP-106”, “HSPP-107”, “HSPP-108”,“HSPP-109”, “HSPP-110”, HSPP-111″, “HSPP-112”, “HSPP-113”, “HSPP-114”,“HSPP-115”, “HSPP-116”, “HSPP-117”, “HSPP-118”, “HSPP-119”, “HSPP-120”,“HSPP-121”, “HSPP-122”, “HSPP-123”, “HSPP-124”, “HSPP-125”, “HSPP-126”,“HSPP-127”, “HSPP-128”, “HSPP-129”, “HSPP-130”, “HSPP-131”, “HSPP-132”,“HSPP-133”, and “HSPP-134”. In one aspect, the invention provides asubstantially purified polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ IDNO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ IDNO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQID NO:132, SEQ ID NO:133, SEQ ID NO:134 (SEQ ID NO:1-134), and fragmentsthereof.

The invention further provides a substantially purified variant havingat least 90% amino acid identity to at least one of the amino acidsequences selected from the group consisting of SEQ ID NO:1-134, andfragments thereof. The invention also provides an isolated and purifiedpolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1-134, andfragments thereof. The invention also includes an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-134, and fragments thereof.

Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1-134, andfragments thereof. The invention also provides an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO:1-134, andfragments thereof.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148,SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ IDNO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162,SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ IDNO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176,SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ IDNO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190,SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ IDNO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204,SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ IDNO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218,SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ IDNO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232,SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ IDNO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ IDNO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260,SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ IDNO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268 (SEQ ID NO:135-268),and fragments thereof. The invention further provides an isolated andpurified polynucleotide variant having at least 90% polynucleotidesequence identity to the polynucleotide sequence selected from the groupconsisting of SEQ ID NO:135-268, and fragments thereof. The inventionalso provides an isolated and purified polynucleotide having a sequencewhich is complementary to the polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:135-268, andfragments thereof.

The invention also provides a method for detecting a polynucleotide in asample containing nucleic acids, the method comprising the steps of (a)hybridizing the complement of the polynucleotide sequence to at leastone of the polynucleotides of the sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide in the sample. In one aspect, the methodfurther comprises amplifying the polynucleotide prior to hybridization.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-134, and fragments thereof. In another aspect, the expressionvector is contained within a host cell.

The invention also provides a method for producing a polypeptide, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideunder conditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1-134, and fragmentsthereof, in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide selected from the group consisting of SEQ ID NO:1-134, andfragments thereof. The invention also provides a purified agonist and apurified antagonist to the polypeptide.

The invention also provides a method for treating or preventing adisorder associated with decreased expression or activity of HSPP, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1-134, and fragmentsthereof, in conjunction with a suitable pharmaceutical carrier.

The invention also provides a method for treating or preventing adisorder associated with increased expression or activity of HSPP, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-134, andfragments thereof.

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows nucleotide and polypeptide sequence identification numbers(SEQ ID NO), clone identification numbers (clone ID), cDNA libraries,and cDNA fragments used to assemble full-length sequences encoding HSPP.

Table 2 shows features of each polypeptide sequence, including predictedsignal peptide sequences, and methods and algorithms used foridentification of HSPP.

Table 3 shows the tissue-specific expression patterns of each nucleicacid sequence as determined by northern analysis, diseases, disorders,or conditions associated with these tissues, and the vector into whicheach cDNA was cloned.

Table 4 describes the tissues used to construct the cDNA libraries fromwhich Incyte cDNA clones encoding HSPP were isolated.

Table 5 shows the programs, their descriptions, references, andthreshold parameters used to analyze HSPP.

Table 6 shows the regions of the full-length nucleotide sequences ofHSPP to which cDNA fragments of Table 1 correspond.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

“HSPP” refers to the amino acid sequences of substantially purified HSPPobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and preferably the humanspecies, from any source, whether natural, synthetic, semi-synthetic, orrecombinant.

The term “agonist” refers to a molecule which, when bound to HSPP,increases or prolongs the duration of the effect of HSPP. Agonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to and modulate the effect of HSPP.

An “allelic variant” is an alternative form of the gene encoding HSPP.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding HSPP include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polynucleotide the same as HSPP or a polypeptide with atleast one functional characteristic of HSPP. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingHSPP, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotidesequence encoding HSPP. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent HSPP. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of HSPP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments,” “immunogenic fragments,” or “antigenicfragments” refer to fragments of HSPP which are preferably at least 5 toabout 15 amino acids in length, most preferably at least 14 amino acids,and which retain some biological activity or immunological activity ofHSPP. Where “amino acid sequence” is recited to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms are not meant to limit the amino acid sequenceto the complete native amino acid sequence associated with the recitedprotein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which, when bound to HSPP,decreases the amount or the duration of the effect of the biological orimmunological activity of HSPP. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, or any other molecules whichdecrease the effect of HSPP.

The term “antibody” refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable ofbinding the epitopic determinant. Antibodies that bind HSPP polypeptidescan be prepared using intact polypeptides or using fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal (e.g., a mouse, a rat, or arabbit) can be derived from the translation of RNA, or synthesizedchemically, and can be conjugated to a carrier protein if desired.Commonly used carriers that are chemically coupled to peptides includebovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin(KLH). The coupled peptide is then used to immunize the animal.

The term “antigenic determinant” refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (given regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition containing a nucleic acidsequence which is complementary to the “sense” strand of a specificnucleic acid sequence. Antisense molecules may be produced by any methodincluding synthesis or transcription. Once introduced into a cell, thecomplementary nucleotides combine with natural sequences produced by thecell to form duplexes and to block either transcription or translation.The designation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

The term “biologically active,” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic HSPP, or of any oligopeptide thereof,to induce a specific immune response in appropriate animals or cells andto bind with specific antibodies.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence“5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A 5′.”Complementarity between two single-stranded molecules may be “partial,”such that only some of the nucleic acids bind, or it may be “complete,”such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encoding HSPPor fragments of HSPP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beenresequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW Fragment Assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

The term “correlates with expression of a polynucleotide” indicates thatthe detection of the presence of nucleic acids, the same or related to anucleic acid sequence encoding HSPP, by northern analysis is indicativeof the presence of nucleic acids encoding HSPP in a sample, and therebycorrelates with expression of the transcript from the polynucleotideencoding HSPP.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term “similarity” refers to a degree of complementarity. There maybe partial similarity or complete similarity. The word “identity” maysubstitute for the word “similarity.” A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to as “substantially similar.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or northern blot, solution hybridization, and the like) underconditions of reduced stringency. A substantially similar sequence orhybridization probe will compete for and inhibit the binding of acompletely similar (identical) sequence to the target sequence underconditions of reduced stringency. This is not to say that conditions ofreduced stringency are such that non-specific binding is permitted, asreduced stringency conditions require that the binding of two sequencesto one another be a specific (i.e., a selective) interaction. Theabsence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementarity(e.g., less than about 30% similarity or identity). In the absence ofnon-specific binding, the substantially similar sequence or probe willnot hybridize to the second non-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, MadisonWis.) which creates alignments between two or more sequences accordingto methods selected by the user, e.g., the clustal method. (See, e.g.,Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustalalgorithm groups sequences into clusters by examining the distancesbetween all pairs. The clusters are aligned pairwise and then in groups.The percentage similarity between two amino acid sequences, e.g.,sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size, and whichcontain all of the elements required for stable mitotic chromosomesegregation and maintenance.

The term “humanized antibody” refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

“Hybridization” refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., Cot or Rot analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” or “addition” refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to the sequence found in thenaturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray” refers to an arrangement of distinctpolynucleotides on a substrate.

The terms “element” or “array element” in a microarray context, refer tohybridizable polynucleotides arranged on the surface of a substrate.

The term “modulate” refers to a change in the activity of HSPP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of HSPP.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, “fragments” refersto those nucleic acid sequences which, comprise a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:135-268,for example, as distinct from any other sequence in the same genome. Forexample, a fragment of SEQ ID NO:135-268 is useful in hybridization andamplification technologies and in analogous methods that distinguish SEQID NO:135-268 from related polynucleotide sequences. A fragment of SEQID NO:135-268 is at least about 15-20 nucleotides in length. The preciselength of the fragment of SEQ ID NO:135-268 and the region of SEQ IDNO:135-268 to which the fragment corresponds are routinely determinableby one of ordinary skill in the art based on the intended purpose forthe fragment. In some cases, a fragment, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to 60 nucleotides, preferably about 15 to 30nucleotides, and most preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or in a hybridization assay or microarray.“Oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining nucleic acids encoding HSPP, or fragments thereof, or HSPPitself, may comprise a bodily fluid; an extract from a cell, chromosome,organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA,or cDNA, in solution or bound to a substrate; a tissue; a tissue print;etc.

The terms “specific binding” or “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, oran antagonist. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide containingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “stringent conditions” refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent, e.g., formamide, temperature, andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

A “substitution” refers to the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

“Transformation” describes a process by which exogenous DNA enters andchanges a recipient cell. Transformation may occur under natural orartificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

A “variant” of HSPP polypeptides refers to an amino acid sequence thatis altered by one or more amino acid residues. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to HSPP. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

THE INVENTION

The invention is based on the discovery of new human signalpeptide-containing proteins (HSPP), the polynucleotides encoding HSPP,and the use of these compositions for the diagnosis, treatment, orprevention of cell proliferative disorders including cancer;inflammation; and cardiovascular, neurological, reproductive, anddevelopmental disorders.

Table 1 lists the Incyte Clones used to derive full length nucleotidesequences encoding HSPP. Columns 1 and 2 show the sequenceidentification numbers (SEQ ID NO) of the amino acid and nucleic acidsequences, respectively. Column 3 shows the Clone ID of the Incyte Clonein which nucleic acids encoding each HSPP were identified, and column 4,the cDNA libraries from which these clones were isolated. Column 5 showsIncyte clones, their corresponding cDNA libraries, and shotgunsequences. The clones and shotgun sequences are part of the consensusnucleotide sequence of each HSPP and are useful as fragments inhybridization technologies.

Table 6 shows the regions of the full-length nucleotide sequences ofHSPP to which cDNA fragments of Table 1 correspond. Column 1 listsnucleotide sequence identifiers and column 2 shows the clone ID of theIncyte clone in which nucleic acids encoding each HSPP were identified.Column 3 shows Incyte clones and shotgun sequences which are part of theconsensus nucleotide sequence of each HSPP and are useful as fragmentsin hybridization technologies. Column 4 lists the starting nucleotideposition and column 5 the ending nucleotide position of the region ofthe full-length HSPP to which the cDNA fragment corresponds.

The columns of Table 2 show various properties of the polypeptides ofthe invention: column 1 references the SEQ ID NO; column 2 shows thenumber of amino acid residues in each polypeptide; column 3, potentialphosphorylation sites; column 4, potential glycosylation sites; column5, the amino acid residues comprising signature sequences and motifs;column 6, the identity of each protein; and column 7, analytical methodsused to identify each HSPP as a signal peptide-containing protein. Notethat in column 5, the first line of each cell lists the amino acidresidues comprising predicted signal peptide sequences. Additionalidentifying motifs or signatures are also listed in column 5. Ofparticular note is the presence of a glycosyl hydrolase family 9 activesite signature in SEQ ID NO:126, a ribosomal protein S18 signature inSEQ ID NO:127, an adrenodoxin family iron-sulfur binding regionsignature and a cytochrome c family heme-binding site signature in SEQID NO:132, and a urotensin II signature sequence in SEQ ID NO:96.

Using BLAST, SEQ ID NO:68 (HSPP-68) has been identified as aTWIK-related acid-sensitive K⁺ channel, and SEQ ID NO:92 (HSPP-92) hasbeen identified as a tyrosine-specific protein phosphatase. Thetyrosine-specific protein phosphatases signature in SEQ ID NO:92(HSPP-92) from about V328 through about F340 (including the putativeactive site cysteine residue at C330) was identified using BLOCKS andPRINTS. Also of note is the identification of SEQ ID NO:66 (HSPP-66) asa steroid binding protein using BLAST.

The columns of Table 3 show the tissue-specificity and diseases,disorders, or conditions associated with nucleotide sequences encodingHSPP. The first column of Table 3 lists the nucleotide sequenceidentifiers. The second column lists tissue categories which expressHSPP as a fraction of total tissue categories expressing HSPP. The thirdcolumn lists the diseases, disorders, or conditions associated withthose tissues expressing HSPP. The fourth column lists the vectors usedto subclone the cDNA library. Of particular note is the expression ofSEQ ID NO:200, SEQ ID NO:203, and SEQ ID NO:225 in lung tissues; theexpression of SEQ ID NO:212, SEQ ID NO:216, and SEQ ID NO:220 inreproductive tissues; the expression of SEQ ID NO:223 in canceroustissues; the expression of SEQ ID NO:232 in gastrointestinal tissue,specifically the small intestine or colon (fifteen out of sixteen(93.8%) cDNA libraries); and the expression of SEQ ID NO:224 incancerous and proliferating tissues. Also of particular interest is thetissue-specific expression of SEQ ID NO:252 and SEQ ID NO:257. SEQ IDNO:252 is derived from OVARTUT01, an ovarian tumor cDNA library and isexclusively expressed in reproductive tumor tissue. SEQ ID NO:257 isderived from THP1AZT01, a 5-aza-2′-deoxycytidine treated humanpromonocyte cDNA library and is exclusively expressed in hematopoietictissue.

The following fragments of the nucleotide sequences encoding HSPP areuseful in hybridization or amplification technologies to identify SEQ IDNO:135-268 and to distinguish between SEQ ID NO:135-268 and relatedpolynucleotide sequences. The useful fragments are the fragment of SEQID NO:230 from about nucleotide 75 to about nucleotide 104; the fragmentof SEQ ID NO:231 from about nucleotide 210 to about nucleotide 239; thefragment of SEQ ID NO:232 from about nucleotide 157 to about nucleotide186; the fragment of SEQ ID NO:233 from about nucleotide 268 to aboutnucleotide 297; the fragment of SEQ ID NO:234 from about nucleotide 160to about nucleotide 186; the fragment of SEQ ID NO:235 from aboutnucleotide 201 to about nucleotide 230; the fragment of SEQ ID NO:236from about nucleotide 165 to about nucleotide 194; the fragment of SEQID NO:237 from about nucleotide 366 to about nucleotide 395; thefragment of SEQ ID NO:238 from about nucleotide 714 to about nucleotide743; the fragment of SEQ ID NO:239 from about nucleotide 1731 to aboutnucleotide 1760; the fragment of SEQ ID NO:240 from about nucleotide 419to about nucleotide 448; the fragment of SEQ ID NO:241 from aboutnucleotide 494 to about nucleotide 523; the fragment of SEQ ID NO:242from about nucleotide 100 to about nucleotide 129; the fragment of SEQID NO:243 from about nucleotide 104 to about nucleotide 133; thefragment of SEQ ID NO:244 from about nucleotide 136 to about nucleotide165; the fragment of SEQ ID NO:245 from about nucleotide 140 to aboutnucleotide 169; the fragment of SEQ ID NO:246 from about nucleotide 125to about nucleotide 154; the fragment of SEQ ID NO:247 from aboutnucleotide 687 to about nucleotide 758; the fragment of SEQ ID NO:248from about nucleotide 327 to about nucleotide 398; the fragment of SEQID NO:249 from about nucleotide 741 to about nucleotide 785; thefragment of SEQ ID NO:250 from about nucleotide 184 to about nucleotide255; the fragment of SEQ ID NO:251 from about nucleotide 165 to aboutnucleotide 242; the fragment of SEQ ID NO:252 from about nucleotide 271to about nucleotide 342; the fragment of SEQ ID NO:253 from aboutnucleotide 1081 to about nucleotide 1152; the fragment of SEQ ID NO:254from about nucleotide 781 to about nucleotide 852; the fragment of SEQID NO:255 from about nucleotide 620 to about nucleotide 691; thefragment of SEQ ID NO:256 from about nucleotide 872 to about nucleotide916; the fragment of SEQ ID NO:257 from about nucleotide 242 to aboutnucleotide 313; the fragment of SEQ ID NO:258 from about nucleotide 595to about nucleotide 648; the fragment of SEQ ID NO:259 from aboutnucleotide 163 to about nucleotide 216; the fragment of SEQ ID NO:260from about nucleotide 244 to about nucleotide 315; the fragment of SEQID NO:261 from about nucleotide 75 to about nucleotide 128; the fragmentof SEQ ID NO:262 from about nucleotide 650 to about nucleotide 703; thefragment of SEQ ID NO:263 from about nucleotide 143 to about nucleotide214; the fragment of SEQ ID NO:264 from about nucleotide 434 to aboutnucleotide 487; the fragment of SEQ ID NO:265 from about nucleotide 218to about nucleotide 271; the fragment of SEQ ID NO:266 from aboutnucleotide 89 to about nucleotide 145; the fragment of SEQ ID NO:267from about nucleotide 198 to about nucleotide 254; and the fragment ofSEQ ID NO:268 from about nucleotide 10 to about nucleotide 54.

The invention also encompasses HSPP variants. A preferred HSPP variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HSPP amino acid sequence, and which contains at least one functionalor structural characteristic of HSPP.

The invention also encompasses polynucleotides which encode HSPP. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:135-268, which encodes HSPP.

The invention also encompasses a variant of a polynucleotide sequenceencoding HSPP. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HSPP. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:135-268 whichhas at least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to anucleic acid sequence selected from the group consisting of SEQ IDNO:135-268. Any one of the polynucleotide variants described above canencode an amino acid sequence which contains at least one functional orstructural characteristic of HSPP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HSPP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HSPP, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HSPP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HSPP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HSPP or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding HSPP and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeHSPP and HSPP derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding HSPP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:135-268 and fragments thereofunder various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) For example, stringent salt concentrationwill ordinarily be less than about 750 mM NaCl and 75 mM trisodiumcitrate, preferably less than about 500 mM NaCl and 50 mM trisodiumcitrate, and most preferably less than about 250 mM NaCl and 25 mMtrisodium citrate. Low stringency hybridization can be obtained in theabsence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Perkin-Elmer),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.),Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown Mass.) andthe ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried outusing either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or theMEGABACE 1000 DNA sequencing system (Molecular Dynamics, SunnyvaleCalif.). The resulting sequences are analyzed using a variety ofalgorithms which are well known in the art. (See, e.g., Ausubel, F. M.(1997) Short Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology andBiotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

The nucleic acid sequences encoding HSPP may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR,Perkin-Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HSPP may be cloned in recombinant DNAmolecules that direct expression of HSPP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express HSPP.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HSPP-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

In another embodiment, sequences encoding HSPP may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, HSPP itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin-Elmer).Additionally, the amino acid sequence of HSPP, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman, New York N.Y.)

In order to express a biologically active HSPP, the nucleotide sequencesencoding HSPP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding HSPP. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding HSPP. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding HSPP and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HSPP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HSPP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding HSPP. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding HSPP can be achieved using amultifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JollaCalif.) or pSPORT1 plasmid (Life Technologies). Ligation of sequencesencoding HSPP into the vector's multiple cloning site disrupts the lacZgene, allowing a colorimetric screening procedure for identification oftransformed bacteria containing recombinant molecules. In addition,these vectors may be useful for in vitro transcription, dideoxysequencing, single strand rescue with helper phage, and creation ofnested deletions in the cloned sequence. (See, e.g., Van Heeke, G. andS. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of HSPP are needed, e.g. for the production of antibodies,vectors which direct high level expression of HSPP may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

Yeast expression systems may be used for production of HSPP. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, 1995, supra; Grant et al. (1987)Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994)Bio/Technology 12:181-184.)

Plant systems may also be used for expression of HSPP. Transcription ofsequences encoding HSPP may be driven viral promoters, e.g., the 35S and19S promoters of CaMV used alone or in combination with the omega leadersequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding HSPP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses HSPP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat. Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of HSPP in cell lines is preferred. For example,sequences encoding HSPP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides, neomycinand G-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP; Clontech), βglucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHSPP is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HSPP can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HSPP under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingHSPP and that express HSPP may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of HSPPusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on HSPPis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St PaulMinn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols inImmunology, Greene Pub. Associates and Wiley-Interscience, New YorkN.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press,Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HSPP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HSPP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HSPP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHSPP may be designed to contain signal sequences which direct secretionof HSPP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Manassas, Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HSPP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric HSPPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of HSPP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His (SEQ ID NO: 269), FLAG, c-myc, and hemagglutinin (HA). GST, MBP,Trx, CBP, and 6-His (SEQ ID NO: 269) enable purification of theircognate fusion proteins on immobilized glutathione, maltose,phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purificationof fusion proteins using commercially available monoclonal andpolyclonal antibodies that specifically recognize these epitope tags. Afusion protein may also be engineered to contain a proteolytic cleavagesite located between the HSPP encoding sequence and the heterologousprotein sequence, so that HSPP may be cleaved away from the heterologousmoiety following purification. Methods for fusion protein expression andpurification are discussed in Ausubel (1995, supra, ch 10). A variety ofcommercially available kits may also be used to facilitate expressionand purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeled HSPPmay be achieved in vitro using the TNT rabbit reticulocyte lysate orwheat germ extract systems (Promega). These systems couple transcriptionand translation of protein-coding sequences operably associated with theT7, T3, or SP6 promoters. Translation takes place in the presence of aradiolabeled amino acid precursor, preferably ³⁵S-methionine.

Fragments of HSPP may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performedby manual techniques or by automation. Automated synthesis may beachieved, for example, using the ABI 431A Peptide Synthesizer(Perkin-Elmer). Various fragments of HSPP may be synthesized separatelyand then combined to produce the full length molecule.

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of HSPP and signal peptide sequences.In addition, chemical and structural similarity, in the context ofsequences and motifs, exists between HSPP-66 and prostaticsteriod-binding C3 precursor from rat (GI 206453); between HSPP-68 andTWIK-related acid-sensitive K+channel from human (GI 2465542); andbetween HSPP-92 and tyrosine specific protein phosphatases (PROSITEPD0000323). In addition, the expression of HSPP is closely associatedwith proliferative, cancerous, inflamed, cardiovascular, nervous,reproductive, hematopoietic/immune, and developmental tissue. Therefore,HSPP appears to play a role in cell proliferative disorders includingcancer; inflammation; and cardiovascular, neurological, reproductive,and developmental disorders. In the treatment of cell proliferativedisorders including cancer; inflammation; and cardiovascular,neurological, reproductive, and developmental disorders associated withincreased HSPP expression or activity, it is desirable to decrease theexpression or activity of HSPP. In the treatment of the above conditionsassociated with decreased HSPP expression or activity, it is desirableto increase the expression or activity of HSPP.

Therefore, in one embodiment, HSPP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of HSPP. Examples ofsuch disorders include, but are not limited to, cell proliferativedisorders such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; inflammatory disorders,such as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma;cardiovascular disorders including disorders of the blood vessels suchas arteriovenous fistula, atherosclerosis, hypertension, vasculitis,Raynaud's disease, aneurysms, arterial dissections, varicose veins,thrombophlebitis and phlebothrombosis, and vascular tumors; disorders ofthe heart such as congestive heart failure, ischemic heart disease,angina pectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, and congenitalheart disease; and disorders of the lungs such as congenital lunganomalies, atelectasis, pulmonary congestion and edema, pulmonaryembolism, pulmonary hemorrhage, pulmonary infarction, pulmonaryhypertension, vascular sclerosis, obstructive pulmonary disease,restrictive pulmonary disease, chronic obstructive pulmonary disease,emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess,pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiliabronchiolitis obliterans-organizing pneumonia, diffuse pulmonaryhemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonaryhemosiderosis, pulmonary involvement in collagen-vascular disorders,pulmonary alveolar proteinosis, lung tumors, inflammatory andnoninflammatory pleural effusions, pneumothorax, and pleural tumors;neurological disorders such as epilepsy, ischemic cerebrovasculardisease, stroke, cerebral neoplasms, Alzheimer's disease, Pick'sdisease, Huntington's disease, dementia, Parkinson's disease and otherextrapyramidal disorders, amyotrophic lateral sclerosis and other motorneuron disorders, progressive neural muscular atrophy, retinitispigmentosa, hereditary ataxias, multiple sclerosis and otherdemyelinating diseases, bacterial and viral meningitis, brain abscess,subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease; prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder; reproductive disorders such as disorders of prolactinproduction; infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, uterine fibroids, autoimmunedisorders, ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia; anddevelopmental disorders, such as renal tubular acidosis, anemia,Cushing's syndrome, achondroplastic dwarfism, Duchenne and Beckermuscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms'tumor, aniridia, genitourinary abnormalities, and mental retardation),Smith-Magenis syndrome, myelodysplastic syndrome, hereditarymucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, and sensorineuralhearing loss.

In another embodiment, a vector capable of expressing HSPP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofHSPP including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HSPP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofHSPP including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofHSPP may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of HSPP including, butnot limited to, those listed above.

In a further embodiment, an antagonist of HSPP may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of HSPP. Examples of such disorders include, butare not limited to, those described above. In one aspect, an antibodywhich specifically binds HSPP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HSPP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HSPP may be administered to a subject to treator prevent a disorder associated with increased expression or activityof HSPP including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HSPP may be produced using methods which are generallyknown in the art. In particular, purified HSPP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HSPP. Antibodies to HSPP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HSPP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HSPP have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HSPP amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to HSPP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell. Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HSPP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HSPP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HSPP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HSPP epitopes is preferred, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for HSPP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of HSPP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple HSPP epitopes, represents the average affinity,or avidity, of the antibodies for HSPP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular HSPP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theHSPP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of HSPP, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington, D.C.; Liddell, J. E. andCryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley& Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of HSPP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingHSPP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHSPP may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HSPP. Thus,complementary molecules or fragments may be used to modulate HSPPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding HSPP.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding HSPP. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

Genes encoding HSPP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding HSPP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHSPP. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHSPP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HSPP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HSPP,antibodies to HSPP, and mimetics, agonists, antagonists, or inhibitorsof HSPP. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HSPP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HSPP or fragments thereof, antibodies of HSPP,and agonists, antagonists or inhibitors of HSPP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HSPP may beused for the diagnosis of disorders characterized by expression of HSPP,or in assays to monitor patients being treated with HSPP or agonists,antagonists, or inhibitors of HSPP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for HSPP include methods which utilizethe antibody and a label to detect HSPP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring HSPP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HSPP expression. Normal or standard values for HSPPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHSPP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HSPP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHSPP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHSPP may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of HSPP, and tomonitor regulation of HSPP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HSPP or closely related molecules may be used to identifynucleic acid sequences which encode HSPP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HSPP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the HSPPencoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ IDNO:135-268 or from genomic sequences including promoters, enhancers, andintrons of the HSPP gene.

Means for producing specific hybridization probes for DNAs encoding HSPPinclude the cloning of polynucleotide sequences encoding HSPP or HSPPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HSPP may be used for the diagnosis ofdisorders associated with expression of HSPP. Examples of such disordersinclude, but are not limited to, cell proliferative disorders such asactinic keratosis, arteriosclerosis, atherosclerosis, bursitis,cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; inflammatory disorders,such as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma;cardiovascular disorders including disorders of the blood vessels suchas arteriovenous fistula, atherosclerosis, hypertension, vasculitis,Raynaud's disease, aneurysms, arterial dissections, varicose veins,thrombophlebitis and phlebothrombosis, and vascular tumors; disorders ofthe heart such as congestive heart failure, ischemic heart disease,angina pectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, and congenitalheart disease; and disorders of the lungs such as congenital lunganomalies, atelectasis, pulmonary congestion and edema, pulmonaryembolism, pulmonary hemorrhage, pulmonary infarction, pulmonaryhypertension, vascular sclerosis, obstructive pulmonary disease,restrictive pulmonary disease, chronic obstructive pulmonary disease,emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess,pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiliabronchiolitis obliterans-organizing pneumonia, diffuse pulmonaryhemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonaryhemosiderosis, pulmonary involvement in collagen-vascular disorders,pulmonary alveolar proteinosis, lung tumors, inflammatory andnoninflammatory pleural effusions, pneumothorax, and pleural tumors;neurological disorders such as epilepsy, ischemic cerebrovasculardisease, stroke, cerebral neoplasms, Alzheimer's disease, Pick'sdisease, Huntington's disease, dementia, Parkinson's disease and otherextrapyramidal disorders, amyotrophic lateral sclerosis and other motorneuron disorders, progressive neural muscular atrophy, retinitispigmentosa, hereditary ataxias, multiple sclerosis and otherdemyelinating diseases, bacterial and viral meningitis, brain abscess,subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease; prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder; reproductive disorders such as disorders of prolactinproduction; infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, uterine fibroids, autoimmunedisorders, ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia; anddevelopmental disorders, such as renal tubular acidosis, anemia,Cushing's syndrome, achondroplastic dwarfism, Duchenne and Beckermuscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms'tumor, aniridia, genitourinary abnormalities, and mental retardation),Smith-Magenis syndrome, myelodysplastic syndrome, hereditarymucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, and sensorineuralhearing loss. The polynucleotide sequences encoding HSPP may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; in dipstick, pin, and multiformatELISA-like assays; and in microarrays utilizing fluids or tissues frompatients to detect altered HSPP expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HSPP may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHSPP may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HSPP in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HSPP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HSPP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HSPP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HSPP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HSPP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HSPPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHSPP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet.15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J.(1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HSPP on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, HSPP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HSPPand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with HSPP, or fragments thereof, and washed. Bound HSPP is thendetected by methods well known in the art. Purified HSPP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HSPP specificallycompete with a test compound for binding HSPP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HSPP.

In additional embodiments, the nucleotide sequences which encode HSPPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The disclosures of all applications, patents, and publications,mentioned above and below, in particular U.S. Ser. No. 60/090,762, U.S.Ser. No. 60/094,983, U.S. Ser. No. 60/102,686, and U.S. Ser. No.60/112,129, are hereby expressly incorporated by reference.

EXAMPLES I. Construction of cDNA Libraries

RNA was purchased from Clontech or isolated from tissues described inTable 4. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL (Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A+) RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Valencia Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See, e.g., Ausubel,1997, supra, units 5.1-6.6). Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), or pINCY (Incyte Corporation, Palo Alto Calif.).Recombinant plasmids were transformed into competent E. coli cellsincluding XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5ÿ, DH10B,or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids were recovered from host cells by in vivo excision, using theUNIZAP vector system (Stratagene) or cell lysis. Plasmids were purifiedusing at least one of the following: a MAGIC or WIZARD minipreps DNApurification system (Promega); an AGTC miniprep purification kit (EdgeBiosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 PlusPlasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep96 plasmid kit from QIAGEN. Following precipitation, plasmids wereresuspended in 0.1 ml of distilled water and stored, with or withoutlyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a Fluoroskan II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

The cDNAs were prepared for sequencing using the ABI CATALYST 800(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) orMICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermalcyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols,base calling software, and kits. In one alternative, cDNAs weresequenced using the MEGABACE 1000 DNA sequencing system (MolecularDynamics). In another alternative, the cDNAs were amplified andsequenced using the ABI PRISM BIGDYE terminator cycle sequencing readyreaction kit (Perkin-Elmer). In yet another alternative, cDNAs weresequenced using solutions and dyes from Amersham Pharmacia Biotech.Reading frames for the ESTs were determined using standard methods(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequenceswere selected for extension using the techniques disclosed in Example V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 5 summarizes the software programs, descriptions, references,and threshold parameters used. The first column of Table 5 shows thetools, programs, and algorithms used, the second column provides a briefdescription thereof, the third column presents the references which areincorporated by reference herein, and the fourth column presents, whereapplicable, the scores, probability values, and other parameters used toevaluate the strength of a match between two sequences (the higher theprobability the greater the homology). Sequences were analyzed usingMACDNASIS PRO software (Hitachi Software Engineering, South SanFrancisco Calif.) and LASERGENE software (DNASTAR).

The polynucleotide sequences were validated by removing vector, linker,and polyA sequences and by masking ambiguous bases, using algorithms andprograms based on BLAST, dynamic programming, and dinucleotide nearestneighbor analysis. The sequences were then queried against a selectionof public databases such as GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,using programs based on BLAST, FASTA, and BLIMPS. The sequences wereassembled into full length polynucleotide sequences using programs basedon Phred, Phrap, and Consed, and were screened for open reading framesusing programs based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length amino acid sequences, and these full length sequences weresubsequently analyzed by querying against databases such as the GenBankdatabases (described above), SwissProt, BLOCKS, PRINTS, Prosite, andHidden Markov Model (HMM)-based protein family databases such as PFAM.HMM is a probalistic approach which analyzes consensus primarystructures of gene families. (See, e.g., Eddy, S. R. (1996) Cur. Opin.Str. Biol. 6:361-365.)

The programs described above for the assembly and analysis of fulllength polynucleotide and amino acid sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:135-268.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies were described in TheInvention section above.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Corporation). This analysis is much fasterthan multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar. The basis ofthe search is the product score, which is defined as:

$\frac{\% \mspace{14mu} {sequence}\mspace{14mu} {identity} \times \% \mspace{14mu} {maximum}\mspace{14mu} {BLAST}\mspace{14mu} {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify relatedmolecules.

The results of northern analyses are reported as a percentagedistribution of libraries in which the transcript encoding HSPPoccurred. Analysis involved the categorization of cDNA libraries byorgan/tissue and disease. The organ/tissue categories includedcardiovascular, dermatologic, developmental, endocrine,gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,reproductive, and urologic. The disease/condition categories includedcancer, inflammation/trauma, cell proliferation, neurological, andpooled. For each category, the number of libraries expressing thesequence of interest was counted and divided by the total number oflibraries across all categories. Percentage values of tissue-specificand disease- or condition-specific expression are reported in Table 3.

V. Extension of HSPP Encoding Polynucleotides

Full length nucleic acid sequences of SEQ ID NOs:135-229 were producedby extension of the component fragments described in Table 1, column 5,using oligonucleotide primers based on these fragments. For each nucleicacid sequence, one primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06(National Biosciences, Plymouth, Minn.), or another appropriate program,to be about 22 to 30 nucleotides in length, to have a GC content ofabout 50% or more, and to anneal to the target sequence at temperaturesof about 68° C. to about 72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations wasavoided.

Selected human cDNA libraries (GIBco BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (The Perkin-Elmer Corp., Norwalk, Conn.) andthoroughly mixing the enzyme and reaction mix. PCR was performed usingthe PTC-200 thermal cycler (MJ Research, Inc., Watertown, Mass.),beginning with 40 pmol of each primer and the recommended concentrationsof all other components of the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

The full length nucleic acid sequences of SEQ ID NO:230-268 wereproduced by extension of an appropriate fragment of the full lengthmolecule using oligonucleotide primers designed from this fragment. Oneprimer was synthesized to initiate 5′ extension of the known fragment,and the other primer, to initiate 3′ extension of the known fragment.The initial primers were designed using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and ÿ-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100 DPICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,Eugene Oreg.) dissolved in 1×TE and 0.5 ÿl of undiluted PCR product intoeach well of an opaque fluorimeter plate (Corning Costar, Acton Mass.),allowing the DNA to bind to the reagent. The plate was scanned in aFluoroskan II (Labsystems Oy, Helsinki, Finland) to measure thefluorescence of the sample and to quantify the concentration of DNA. A 5μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose mini-gel to determine which reactionswere successful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2× carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulphoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Perkin-Elmer).

In like manner, the nucleotide sequences of SEQ ID NO:135-268 are usedto obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for such extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:135-268 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [ÿ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, XbaI,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots to film forseveral hours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the HSPP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HSPP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of HSPP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the HSPP-encoding transcript.

IX. Expression of HSPP

Expression and purification of HSPP is achieved using bacterial orvirus-based expression systems. For expression of HSPP in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express HSPP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof HSPP in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding HSPP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, HSPP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from HSPP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified HSPP obtained by these methods can beused directly in the following activity assay.

X. Demonstration of HSPP Activity

HSPP-68

HSPP-68 activity is measured by determining the potassium current usingvoltage clamp analysis on single Xenopus laevis oocytes injected withHSPP-68 cRNA. HSPP-68 cRNA is synthesized in vitro from linearizedHSPP-68 encoding plasmids using the T7 RNA polymerase and injected intooocytes. Injected oocytes are used two to four days after injection. Ina 0.3 ml perfusion chamber, a single oocyte is impaled with two standardmicroelectrodes (1-2.5 Mÿ) filled with 3 M KCl. The oocyte is maintainedunder voltage clamp by using a Dagan TEV 200 amplifier, in buffercontaining 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES,pH 7.4 with NaOH. Stimulation of the preparation, data acquisition, andanalysis is performed using a computer. All experiments are performed atroom temperature (21-22° C.). Following a depolarizing pulse, thecharacteristics of the resulting potassium current are measured via therecording electrode. The amount of potassium current that flows inresponse to a unit depolarization is proportional to the activity ofHSPP-68 in the cell. (Duprat, F. et al. (1997) EMBO J. 16:5464-5471.)

HSPP-92

HSPP-92 protein phosphatase activity is measured by the hydrolysis ofP-nitrophenyl phosphate (PNPP). HSPP-92 is incubated together with PNPPin HEPES buffer pH 7.5, in the presence of 0.1% b-mercaptoethanol at 37°C. for 60 min. The reaction is stopped by the addition of 6 ml of 10 NNaOH and the increase in light absorbance at 410 nm resulting from thehydrolysis of PNPP is measured using a spectrophotometer. The increasein light absorbance is proportional to the activity of PP in the assay.(Diamond R. H. et al (1994) Mol Cell Biol 14:3752-62.)

Alternatively, HSPP, or biologically active fragments thereof, arelabeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al.(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed inthe wells of a multi-well plate are incubated with the labeled HSPP,washed, and any wells with labeled HSPP complex are assayed. Dataobtained using different concentrations of HSPP are used to calculatevalues for the number, affinity, and association of HSPP with thecandidate molecules.

Alternatively, an assay for HSPP activity measures the expression ofHSPP on the cell surface. cDNA encoding HSPP is subcloned into anappropriate mammalian expression vector suitable for high levels of cDNAexpression. The resulting construct is transfected into a nonhuman cellline such as NIH3T3. Cell surface proteins are labeled with biotin usingmethods known in the art. Immunoprecipitations are performed usingHSPP-specific antibodies, and immunoprecipitated samples are analyzedusing SDS-PAGE and immunoblotting techniques. The ratio of labeledimmunoprecipitant to unlabeled immunoprecipitant is proportional to theamount of HSPP expressed on the cell surface.

Alternatively, an assay for HSPP activity measures the amount of HSPP insecretory, membrane-bound organelles. Transfected cells as describedabove are harvested and lysed. The lysate is fractionated using methodsknown to those of skill in the art, for example, sucrose gradientultracentrifugation. Such methods allow the isolation of subcellularcomponents such as the Golgi apparatus, ER, small membrane-boundvesicles, and other secretory organelles. Immunoprecipitations fromfractionated and total cell lysates are performed using HSPP-specificantibodies, and immunoprecipitated samples are analyzed using SDS-PAGEand immunoblotting techniques. The concentration of HSPP in secretoryorganelles relative to HSPP in total cell lysate is proportional to theamount of HSPP in transit through the secretory pathway.

XI. Functional Assays

HSPP function is assessed by expressing the sequences encoding HSPP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. 1-2 μg of an additional plasmidcontaining sequences encoding a marker protein are co-transfected.Expression of a marker protein provides a means to distinguishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York N.Y.

The influence of HSPP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding HSPPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding HSPP and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XII. Production of HSPP Specific Antibodies

HSPP substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the HSPP amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anABI 431 A Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry andcoupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity by, for example,binding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XIII. Purification of Naturally Occurring HSPP Using Specific Antibodies

Naturally occurring or recombinant HSPP is substantially purified byimmunoaffinity chromatography using antibodies specific for HSPP. Animmunoaffinity column is constructed by covalently coupling anti-HSPPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing HSPP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HSPP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HSPP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HSPPis collected.

XIV. Identification of Molecules Which Interact with HSPP

HSPP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HSPP, washed, and anywells with labeled HSPP complex are assayed. Data obtained usingdifferent concentrations of HSPP are used to calculate values for thenumber, affinity, and association of HSPP with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

TABLE 1 Protein SEQ Nucleotide ID NO: SEQ ID NO: Clone ID LibraryFragments 1 135 443531 MPHGNOT03 443531H1 (MPHGNOT03), 1406807F6(LATRTUT02), 443531T6 (MPHGNOT03), SBBA00451F1, SBBA00676F1 2 136 632860NEUTGMT01 632860H1 (NEUTGMT01), 784715R3 (PROSNOT05), 509590H1(MPHGNOT03) 3 137 670010 CRBLNOT01 670010H1 (CRBLNOT01), 669971R1(CRBLNOT01), 1553045F1 (BLADTUT04) 4 138 726498 SYNOOAT01 726498H1(SYNOOAT01), 726498R6 (SYNOOAT01), 866599R3 (BRAITUT03) 5 139 795064OVARNOT03 795064H1 (OVARNOT03), 4339458H1 (BRAUNOT02), 937605R3(CERVNOT01), 2381151F6 (ISLTNOT01), 1466346F6 (PANCTUT02) 6 140 924925BRAINOT04 924925H1 (BRAINOT04), 3268330H1 (BRAINOT20), 759120R3(BRAITUT02) 7 141 962390 BRSTTUT03 962390H1 (BRSTTUT03), 1907958F6(CONNTUT01), 023569F1 (ADENINB01), 167282F1 (LIVRNOT01), 1309211F1(COLNFET02), SAUA00696F1, SAUA02860F1 8 142 1259405 MENITUT03 1259405H1(MENITUT03), 2472425H1 (THP1NOT03), 774303R1 (COLNNOT05), 1520779F1(BLADTUT04), 1693833F6 (COLNNOT23), 1831858T6.comp (THP1AZT01),1527737T6.comp (UCMCL5T01) 9 143 1297384 BRSTNOT07 1297384H1(BRSTNOT07), 1269310F6 (BRAINOT09), 1457367F1 (COLNFET02), 415587R1(BRSTNOT01), SANA02967F1 10 144 1299627 BRSTNOT07 1299627H1 (BRSTNOT07),1359140F6 (LUNGNOT09), 1349224F1 (LATRTUT02), SBAA01431F1, SBAA02909F1,SBAA01156F1 11 145 1306026 PLACNOT02 1306026H1 (PLACNOT02), 1464088R6(PANCNOT04), SBAA02496F1, SBAA04305F1 12 146 1316219 BLADTUT02 1316219H1(BLADTUT02), 2458603F6 (ENDANOT01), 2504756T6 (CONUTUT01) 13 147 1329031PANCNOT07 1329031H1 (PANCNOT07), 1329031T6 (PANCNOT07), 1329031F6(PANCNOT07) 14 148 1483050 CORPNOT02 1483050H1 (CORPNOT02), 855049H1(NGANNOT01), 077017F1 (SYNORAB01), 1483050F6 (CORPNOT02), 1480024T6(CORPNOT02), 1483050T6 (CORPNOT02), 759486R1 (BRAITUT02) 15 149 1514160PANCTUT01 1514160H1 (PANCTUT01), 1866765T7 (SKINBIT01), 782676R1(MYOMNOT01), 008055X4 (HMC1NOT01), 008055X5 (HMC1NOT01), 1866765F6(SKINBIT01), SAOA03127F1 16 150 1603403 LUNGNOT15 1603403H1 (LUNGNOT15),372910F1 (LUNGNOT02), 733299R7 (LUNGNOT03) 17 151 1652303 PROSTUT081652303H1 (PROSTUT08), 1671806H1 (BLADNOT05), 1341743T1 (COLNTUT03),3803812H1 (BLADTUT03), 1878546F6 (LEUKNOT03), 1428640F1 (SINTBST01),2058609R6 (OVARNOT03), 1331621F1 (PANCNOT07), 1306331T1 (PLACNOT02) 18152 1693358 COLNNOT23 1693358H1 (COLNNOT23), 2498265H1 (ADRETUT05),1867125F6 (SKINBIT01), 1693358T6 (COLNNOT23), 2245848R6 (HIPONON02) 19153 1707711 DUODNOT02 1707711H1 (DUODNOT02), 1484609T1 (CORPNOT02),1707711F6 (DUODNOT02), 1267959F1 (BRAINOT09), 1484609F1 (CORPNOT02),SAJA00930F1, SAJA01300R1, SAJA00999R1 20 154 1738735 COLNNOT22 1738735H1(COLNNOT22), SAJA00944R1, SAJA00137F1, SAJA03629F1 21 155 1749147STOMTUT02 1749147H1 (STOMTUT02), 1749147F6 (STOMTUT02), 1749147T6(STOMTUT02) 22 156 1817722 PROSNOT20 1817722H1 (PROSNOT20), 2011085H1(TESTNOT03) 23 157 1831290 THP1AZT01 1831290H1 (THP1AZT01), 3473958H1(LUNGNOT27), 1972268F6 (UCMCL5T01), 1301277F1 (BRSTNOT07), 1521574F1(BLADTUT04), 1561690T6 (SPLNNOT04), 891461R1 (STOMTUT01) 24 158 1831477THP1AZT01 1831477H1 (THP1AZT01), 1582867H1 (DUODNOT01), 1336769T1(COLNNOT13), 1933092H1 (COLNNOT16), 1519909F1 (BLADTUT04), 1220946H1(NEUTGMT01), 809556T1 (LUNGNOT04), 1217559T1 (NEUTGMT01), 1309225F1(COLNFET02) 25 159 1841607 COLNNOT07 1841607H1 (COLNNOT07), SBHA03588F126 160 1852391 LUNGFET03 1852391H1 (LUNGFET03), 734140H1 (TONSNOT01),1852391F6 (LUNGFET03) 27 161 1854555 HNT3AZT01 1854555H1 (HNT3AZT01),2511711H1 (CONUTUT01), 782453R1 (MYOMNOT01), 1854555F6 (HNT3AZT01),1840675T6 (COLNNOT07), 2109736H1 (BRAITUT03) 28 162 1855755 PROSNOT181855755H1 (PROSNOT18), 3040236H1 (BRSTNOT16), 1283207F1 (COLNNOT16),833763T1 (PROSNOT07), 1920926R6 (BRSTTUT01) 29 163 1861434 PROSNOT191861434H1 (PROSNOT19), 980291R1 (TONGTUT01), 1861434T6 (PROSNOT19),SARA01525F1, SARA02548F1 30 164 1872334 LEUKNOT02 1872334H1 (LEUKNOT02),1872334F6 (LEUKNOT02), SBGA03684F1 31 165 1877230 LEUKNOT03 1877230H1(LEUKNOT03), 2519841H1 (BRAITUT21), 1877230T6 (LEUKNOT03), 1254693F1(LUNGFET03), 077020R1 (SYNORAB01), 1232336F1 (LUNGFET03), 1004952R6(BRSTNOT03), SARA01879F1, SARA02654F1 32 166 1877885 LEUKNOT03 1877885H1(LEUKNOT03), 508020F1 (TMLR3DT01), 2751126R6 (THP1AZS08), SARA02571F1 33167 1889269 BLADTUT07 1889269H1 (BLADTUT07), 1915551H1 (PROSTUT04),629493X12 (KIDNNOT05), 1441289F1 (THYRNOT03), 1215274X34F1 (BRSTTUT01),1818447F6 (PROSNOT20), 1208463R1 (BRSTNOT02) 34 168 1890243 BLADTUT071890243H1 (BLADTUT07), SARA01884F1, SATA00046F1, SARA03294F1,SARA02790F1 35 169 1900433 BLADTUT06 1900433H1 (BLADTUT06), SATA00396F1,SATA02742F1 36 170 1909441 CONNTUT01 1909441H1 (CONNTUT01), 1398811F1(BRAITUT08), 3039939H1 (BRSTNOT16), 3324740H1 (PTHYNOT03), 1442131F6(THYRNOT03), 2254056H1 (OVARTUT01), 2199453T6 (SPLNFET02), 1692610F6(COLNNOT23), 1698531H1 (BLADTUT05) 37 171 1932226 COLNNOT16 1932226H1(COLNNOT16), 2320569H1 (OVARNOT02), 1932226F6 (COLNNOT16), 2469455T6(THP1NOT03), 2469455F6 (THP1NOT03), 1907140F6 (OVARNOT07), SATA02592F138 172 1932647 COLNNOT16 1932647H1 (COLNNOT16), 1492745T1 (PROSNON01),1492745H1 (PROSNON01), SASA02355F1, SASA00117F1, SASA00192F1 39 1732124245 BRSTNOT07 2124245H1 (BRSTNOT07), 1235393F1 (LUNGFET03),1402264F6 (LATRTUT02), 1303990F1 (PLACNOT02), 1402264T6 (LATRTUT02) 40174 2132626 OVARNOT03 2132626H1 (OVARNOT03), 1723432T6 (BLADNOT06),2132626R6 (OVARNOT03), 1736723T6 (COLNNOT22), 1504738F1 (BRAITUT07) 41175 2280639 PROSNON01 2280639H1 (PROSNON01), 1435330H1 (PANCNOT08),1377560F6 (LUNGNOT10) 42 176 2292356 BRAINON01 2292356H1 (BRAINON01),4086827H1 (LIVRNOT06), 1754442F6 (LIVRTUT01), 3571126H1 (HEAPNOT01),1601305F6 (BLADNOT03) 43 177 2349310 COLSUCT01 2349310H1 (COLSUCT01),2349310T6 (COLSUCT01) 44 178 2373227 ADRENOT07 2373227H1 (ADRENOT07),3316444H1 (PROSBPT03), 302685R6 (TESTNOT04), SASA02181F1, SASA01923F1,SASA03516F1 45 179 2457682 ENDANOT01 2457682H1 (ENDANOT01), 2457682F6(ENDANOT01) 46 180 2480426 SMCANOT01 2480426H1 (SMCANOT01), 2480426F6(SMCANOT01) 47 181 2503743 CONUTUT01 2503743H1 (CONUTUT01), 1853909H1(HNT3AZT01), 1517619F1 (PANCTUT01), 1467896F6 (PANCTUT02), 490031F1(HNT2AGT01), 1208654R1 (BRSTNOT02), 880544R1 (THYRNOT02) 48 182 2537684BONRTUT01 2537684H1 (BONRTUT01), 2005493H1 (TESTNOT03), 730969H1(LUNGNOT03), 2537601F6 (BONRTUT01), 916487H1 (BRSTNOT04), 996135R1(KIDNTUT01), 1920738R6 (BRSTTUT01), 1957710F6 (CONNNOT01) 49 183 2593853OVARTUT02 2593853H1 (OVARTUT02), 807497H1 (STOMNOT02), 914020R6(STOMNOT02), 889992R1 (STOMTUT01) 50 184 2622354 KERANOT02 2622354H1(KERANOT02), 2623992H1 (KERANOT02), 1556510F6 (BLADTUT04) 51 185 2641377LUNGTUT08 2641377H1 (LUNGTUT08), 4341415H2 (BRAUNOT02), SBCA07049F3 52186 2674857 KIDNNOT19 2674857H1 (KIDNNOT19), 1872373H1 (LEUKNOT02),470512R6 (MMLR1DT01), 1728547H1 (PROSNOT14), 3013651F6 (MUSCNOT07),SBCA01366F1, SBCA00694F1 53 187 2758485 THP1AZS08 2758485H1 (THP1AZS08),3097533H1 (CERVNOT03), 1578959F6 (DUODNOT01) 54 188 2763296 BRSTNOT122763296H1 (BRSTNOT12), 3486025F6 (KIDNNOT31), SBDA07002F3 55 189 2779436OVARTUT03 2779436H1 (OVARTUT03), 2779436F6 (OVARTUT03), SBDA07009F3 56190 2808528 BLADTUT08 2808528H1 (BLADTUT08), 2611513F6 (THYMNOT04),SBDA07021T3 57 191 2809230 BLADTUT08 2809230H1 (BLADTUT08), 2213849H1(SINTFET03), 711706R6 (SYNORAT04), 958323R1 (KIDNNOT05), 030732F1(THP1NOB01) 58 192 2816821 BRSTNOT14 2816821H1 (BRSTNOT14), 3746964H1(THYMNOT08), 2816821F6 (BRSTNOT14), 948722T6 (PANCNOT05), 807947R6(STOMNOT02) 59 193 2817268 BRSTNOT14 2817268H1 (BRSTNOT14), 3591308H1(293TF5T01), 419522R1 (BRSTNOT01), 2073028F6 (ISLTNOT01), 1308781F6(COLNFET02) 60 194 2923165 SININOT04 2923165H1 (SININOT04), 2011630H1(TESTNOT03), 1457250F1 (COLNFET02), 754668R1 (BRAITUT02), 1406510F6(LATRTUT02) 61 195 2949822 KIDNFET01 2949822H1 (KIDNFET01), SBDA07078F362 196 2992192 KIDNFET02 2992192H1 (KIDNFET02), 2534324H2 (BRAINOT18),2815255T6 (OVARNOT10), 1551107T6 (PROSNOT06), 1551107R6 (PROSNOT06) 63197 2992458 KIDNFET02 2992458H1 (KIDNFET02), 2618951H1 (GBLANOT01),1479252F1 (CORPNOT02), 1879054H1 (LEUKNOT03), 1879054F6 (LEUKNOT03),2215240H1 (SINTFET03), 1535968T1 (SPLNNOT04) 64 198 3044710 HEAANOT013044710H1 (HEAANOT01), 3741773H1 (MENTNOT01), 859906X42C1 (BRAITUT03),1534347F1 (SPLNNOT04), 1421122F1 (KIDNNOT09), 1303865F1 (PLACNOT02),1704452F6 (DUODNOT02), 1251642F1 (LUNGFET03), 1781694R6 (PGANNON02) 65199 3120415 LUNGTUT13 3120415H1 (LUNGTUT13), 1360123T1 (LUNGNOT12),1375015H1 (LUNGNOT10) 66 200 126758 LUNGNOT01 126758H1 (LUNGNOT01),126758X11 (LUNGNOT01), 811864T1 (LUNGNOT04) 67 201 674760 CRBLNOT01674760H1 (CRBLNOT01), 3253976H1 (OVARTUN01), SAUA03387F1 68 202 1229438BRAITUT01 1229438H1 (BRAITUT01), 1230616H1 (BRAITUT01), 1461187R1(PANCNOT04), 2493039H1 (ADRETUT05), 2891628H1 (LUNGFET04) 69 203 1236935LUNGFET03 1236935H1 (LUNGFET03), SBAA00983F1, SBAA02057F1, SBAA00170F170 204 1359283 LUNGNOT12 1359283H1 (LUNGNOT12), SBAA01213F1, SBAA03934F171 205 1450703 PENITUT01 551298F1 (BEPINOT01), 551298R1 (BEPINOT01),1450703H1 (PENITUT01), 2748715H1 (LUNGTUT11) 72 206 1910668 CONNTUT011269346H1 (BRAINOT09), 1380872F1 (BRAITUT08), 1910668F6 (CONNTUT01),1910668H1 (CONNTUT01), SATA02800F1, SATA03799F1, SARA02035F1 73 2071955143 CONNNOT01 1955143F6 (CONNNOT01), 1955143H1 (CONNNOT01) 74 2081961637 BRSTNOT04 867025H1 (BRAITUT03), 1961637H1 (BRSTNOT04), 2809064T6(BLADTUT08), 2938714H1 (THYMFET02), 2956402H1 (KIDNFET01), 3808735T6(CONTTUT01) 75 209 1990762 CORPNOT02 1990762H1 (CORPNOT02), 1990762T3(CORPNOT02), SBGA04911F1, SBGA01201F1, SBGA02205F1 76 210 1994131CORPNOT02 1994131H1 (CORPNOT02), 2645984F6 (OVARTUT04) 77 211 1997745BRSTTUT03 1752307F6 (LIVRTUT01), 1853730H1 (HNT3AZT01), 1997745H1(BRSTTUT03), SAZA00953F1 78 212 2009035 TESTNOT03 2009035H1 (TESTNOT03),2009035R6 (TESTNOT03) 79 213 2009152 TESTNOT03 2009152H1 (TESTNOT03),2009152R6 (TESTNOT03), 2783263H1 (BRSTNOT13) 80 214 2061752 OVARNOT032061752H1 (OVARNOT03), 2061752T6 (OVARNOT03), 2732805H1 (OVARTUT04),SAZA01310F1, SAZA00830F1 81 215 2061933 OVARNOT03 046580R1 (CORNNOT01),746061R1 (BRAITUT01), 826996R1 (PROSNOT06), 2061933H1 (OVARNOT03) 82 2162081422 UTRSNOT08 2081422F6 (UTRSNOT08), 2081422H1 (UTRSNOT08),SBCA04793F1, SBCA05657F1, SBDA00065F1 83 217 2101278 BRAITUT02 2101278H1(BRAITUT02), SAXA00399F1, SAXA01284F1, SAXA01227F1 84 218 2121353BRSTNOT07 341437H1 (NEUTFMT01), 687136H1 (UTRSNOT02), 2121353H1(BRSTNOT07), SASA01311F1 85 219 2241736 PANCTUT02 833263H1 (PROSTUT04),2241736H1 (PANCTUT02), SAZA01148F1, SASA03299F1, SASA01349F1 86 2202271935 PROSNON01 2271935H1 (PROSNON01), 2276774H1 (PROSNON01),2760171T6 (THP1AZS08) 87 221 2295344 BRSTNOT05 2295344H1 (BRSTNOT05),3288561F6 (BONRFET01), SBGA01801F1 88 222 2303994 BRSTNOT05 905482T1(COLNNOT08), 1858636F6 (PROSNOT18), 2303994H1 (BRSTNOT05) 89 223 2497805ADRETUT05 2497805F6 (ADRETUT05), 2497805H1 (ADRETUT05) 90 224 2646362LUNGTUT11 1754702H1 (LIVRTUT01), 2640776T6 (LUNGTUT08), 2646362H1(LUNGTUT11), 3356773H1 (PROSTUT16) 91 225 2657146 LUNGTUT09 2657146F6(LUNGTUT09), 2657146H1 (LUNGTUT09) 92 226 2755786 THP1AZS08 288436R1(EOSIHET02), 1252824F6 (LUNGFET03), 1305549H1 (PLACNOT02), 1364975R1(SCORNON02), 2018293H1 (THP1NOT01), 2047320H1 (THP1T7T01), 2184537F6(SININOT01), 2755786H1 (THP1AZS08), 4111022H1 (PROSBPT07) 93 227 2831245TLYMNOT03 2831245H1 (TLYMNOT03), SBMA01396F1 94 228 3116250 LUNGTUT13126263F1 (LUNGNOT01), 2729942H1 (OVARTUT04), 3116250H1 (LUNGTUT13) 95229 3129630 LUNGTUT12 3129630F6 (LUNGTUT12), 3129630H1 (LUNGTUT12),SBDA06436F1 96 230 007632 HMC1NOT01 007632H1 (HMC1NOT01), 007632R6(HMC1NOT01), 007632T6 (HMC1NOT01) 97 231 1236968 LUNGFET03 1236968H1(LUNGFET03), SBAA02713F1, SBAA03203F1, SBAA04196F1 98 232 1334153COLNNOT13 776410R1 (COLNNOT05), 1334153H1 (COLNNOT13), 1334153T1(COLNNOT13), 1800085F6 (COLNNOT27), 2701948H1 (OVARTUT10) 99 233 1396975BRAITUT08 864113H1 (BRAITUT03), 876139R1 (LUNGAST01), 1268313F1(BRAINOT09), 1351348T1 (LATRTUT02), 1396975H1 (BRAITUT08), 1485768F6(CORPNOT02), 1815364F6 (PROSNOT20) 100 234 1501749 SINTBST01 079080R1(SYNORAB01), 1501749H1 (SINTBST01), 1724970H1 (PROSNOT14) 101 2351575240 LNODNOT03 081858R1 (SYNORAB01), 1575240H1 (LNODNOT03), 3451462R6(UTRSNON03) 102 236 1647884 PROSTUT09 1647884H1 (PROSTUT09), 1647884T6(PROSTUT09), 3998922R6 (HNT2AZS07) 103 237 1661144 BRSTNOT09 720941X17(SYNOOAT01), 1661144H1 (BRSTNOT09), 2181782H1 (SININOT01) 104 2381685409 PROSNOT15 755203R1 (BRAITUT02), 1226185T1 (COLNNOT01), 1300837F1(BRSTNOT07), 1685409H1 (PROSNOT15), 1705256H1 (DUODNOT02) 105 2391731419 BRSTTUT08 1731419H1 (BRSTTUT08), 1731419X319T3 (BRSTTUT08),1731419X322F1 (BRSTTUT08), 1731419X326F1 (BRSTTUT08), 1731419X329F1(BRSTTUT08), 1733786F6 (BRSTTUT08), SZAH01494F1 106 240 2650265BRSTNOT14 1680316T6 (STOMFET01), 2650265H1 (BRSTNOT14), 2650265T6(BRSTNOT14), 2760588R6 (BRAINOS12) 107 241 2677129 KIDNNOT19 1592129H1(CARGNOT01), 2645962H1 (OVARTUT04), 2677129F6 (KIDNNOT19), 2677129H1(KIDNNOT19), 2910973H1 (KIDNTUT15), 4571722H1 (PROSTMT02), 4906791H2(TLYMNOT08) 108 242 3151073 ADRENON04 3150857T6 (ADRENON04), 3151073H1(ADRENON04), 3151073R6 (ADRENON04) 109 243 3170095 BRSTNOT18 3170095F6(BRSTNOT18), 3170095H1 (BRSTNOT18) 110 244 3475168 LUNGNOT27 079680F1(SYNORAB01), 443811T6 (MPHGNOT03), 1509356T6 (LUNGNOT14), 1873596F6(LEUKNOT02), 2440867H1 (EOSITXT01), 3475168H1 (LUNGNOT27) 111 2453836893 DENDTNT01 446637H1 (MPHGNOT03), 1219376R6 (NEUTGMT01), 3735467F6(SMCCNOS01), 3735467T6 (SMCCNOS01), 3836893H1 (DENDTNT01) 112 2464072159 KIDNNOT26 2129415T6 (KIDNNOT05), 4072159F6 (KIDNNOT26),4072159H1 (KIDNNOT26) 113 247 1003916 BRSTNOT03 620937R6 (PGANNOT01),1003916H1 and 1003916R6 (BRSTNOT03), 1413623H1 (BRAINOT12), 1435945F1(PANCNOT08), 1479127F1 (CORPNOT02), 1969146R6 (BRSTNOT04), 2517587F6(BRAITUT21), 2967848H1 (SCORNOT04) 114 248 2093492 PANCNOT04 489651H1(HNT2AGT01), 1265353T1 (SYNORAT05), 1431505R6 (BEPINON01), 1605237F6(LUNGNOT15), 2093492H1 and 2093492T6 (PANCNOT04), 4195560H1 (COLITUT02)115 249 2108789 BRAITUT03 2108789H1 and 2108789R6 (BRAITUT03), 2182008T6(SININOT01), 3255751R6 and 3255751T6 (OVARTUN01) 116 250 2171401ENDCNOT03 037241F1 (HUVENOB01), 1821492F6 (GBLATUT01), 2055814T6(BEPINOT01), 2171401F6 and 2171401H1 (ENDCNOT03), 2668952F6 (ESOGTUT02),3140313H1 and 3140313T6 (SMCCNOT02), 5031775H1 (EPIBTXT01) 117 2512212530 SINTFET03 187596R6 and 187596T6 (CARDNOT01), 919634R6(RATRNOT02), 1992331H1 (CORPNOT02), 2062034H1 (OVARNOT03), 2212530F6 and2212530H1 (SINTFET03), 2520479H1 (BRAITUT21), 2878284F6 (THYRNOT10),2992354H1 (KIDNFET02), 4020719F6 (BRAXNOT02) 118 252 2253036 OVARTUT012253036H1 and 2253036R6 (OVARTUT01) 119 253 2280161 PROSNON01 482326H1(HNT2RAT01), 934345H1 (CERVNOT01), 1379358F1 and 1379358T1 (LUNGNOT10),1438562T1 (PANCNOT08), 1467511F6 (PANCTUT02), 1568138F1 (UTRSNOT05),1636106T6 (UTRSNOT06), 2134534F6 (ENDCNOT01), 2280161H1 and 2280161X19F1(PROSNON01), 2789845F6 (COLNTUT16), 3096938H1 (CERVNOT03), 3774621F6(BRSTNOT25), 4222971H1 (PANCNOT07), 5111983H1 (ENDITXT01), 5324177H1(FIBPFEN06) 120 254 2287485 BRAINON01 1454588F1 (PENITUT01), 1593332F6(BRAINOT14), 2287485H1 and 2287485R6 (BRAINON01), 3765992H1 (BRSTNOT24),4374293H1 (CONFNOT03), 4937931H1 (PROSTUS18), SBCA01722F1 121 2552380344 ISLTNOT01 2380344F6 and 2380344H1 (ISLTNOT01), 2888536T3(LUNGFET04), SASA03644F1, SASA03689F1 122 256 2383171 ISLTNOT01 956296R1(KIDNNOT05), 1342250F1 (COLNTUT03), 1468046F1 and 1468046T1 (PANCTUT02),2383171H1 (ISLTNOT01), SBYA05452U1, SBYA01369U1 123 257 2396046THP1AZT01 2396046F6, 2396046H1 and 2396118T6 (THP1AZT01) 124 258 2456587ENDANOT01 2456587H1 and 2456587T6 (ENDANOT01), 2872569H1 (THYRNOT10),SBCA03778F1, SBDA00115F1, SBCA02401F1, SBCA03351F1, SBCA05164F1,SBCA04783F1, SBCA00155F1, SBCA04141F1 125 259 2484813 BONRTUT011234970T1 (LUNGFET03), 1338090F6 (COLNNOT13), 2484813H1 (BONRTUT01),SBCA00053F1, SBCA02064F1, SBCA02151F1, SBCA03770F1, SBCA04866F1,SBCA03406F1 126 260 2493851 ADRETUT05 2493851H1 (ADRETUT05), 3805916F6(BLADTUT03), 4500439H1 and 4500748H1 (BRAVTXT02), 5120601H1 (SMCBUNT01)127 261 2495719 ADRETUT05 603447R1 (BRSTTUT01), 2495719H1 (ADRETUT05),2917493F6 (THYMFET03), 4647103H1 (PROSTUT20), SBRA04984D1 128 2622614153 GBLANOT01 1833135R6 (BRAINON01), 1966515R6 (BRSTNOT04),2331103R6 (COLNNOT11), 2614153H1 (GBLANOT01), 2656691F6 (LUNGTUT09),3951176H1 (DRGCNOT01) 129 263 2655184 THYMNOT04 2655184H1 (THYMNOT04),SBDA05215F1, SBDA05213F1, SBDA01516F1 130 264 2848362 BRSTTUT131297974F1 and 1297974T6 (BRSTNOT07), 2630138F6 (COLNTUT15), 2848362H1(BRSTTUT13) 131 265 2849906 BRSTTUT13 1541617R1 and 1541617T1(SINTTUT01), 2684504F6 and 2684504T6 (LUNGNOT23), 2796805H1 (NPOLNOT01),2849906H1 (BRSTTUT13) 132 266 2899137 DRGCNOT01 2899137H1 (DRGCNOT01),3026490F6 and 3026490T6 (HEARFET02), 3483359H1 (KIDNNOT31) 133 2672986229 CARGDIT01 1740227T6 (HIPONON01), 2986229H1 (CARGDIT01) 134 2683222081 COLNNON03 1754079F6 (LIVRTUT01), 3222081H1 (COLNNON03),4053813T6 (SPLNNOT13), 4230282H1 (BRAMDIT01), SBDA07029F3

TABLE 2 Protein Amino SEQ Acid Potential ID Resi- PotentialGlycosylation Analytical NO: dues Phosphorylation Sites Sites SignatureSequences Identification Methods 1 88 T83 S38 T76 M1-A21 Signal PeptideHMM 2 128 S30 S40 T47 T119 M1-F28 Signal Peptide W125 HMM 3 111 T70M1-T18 Signal Peptide HMM 4 110 S32 T64 N58 M1-A29 Signal Peptide HMM 578 T27 S39 S39 S44 S22 M1-R24 Signal Peptide T27 S28 S57 HMM 6 88 T55S30 S40 T55 N34 M1-N21 Signal Peptide HMM 7 227 S220 S70 S83 T131 N100M1-Q20 Signal Peptide S134 S141 T158 HMM Y123 8 198 S62 T123 S142 S189N60 M1-A28 Signal Peptide S62 T100 Y85 HMM 9 65 T48 M1-A29 SignalPeptide HMM 10 154 M1-A29 Signal Peptide HMM 11 237 T116 T26 T79 T85N128 M1-A19 Signal Peptide T182 T188 T194 HMM T206 S60 S123 S176 S213 12225 T158 S128 N166 M1-G27 Signal Peptide HMM 13 117 S41 M1-A23 SignalPeptide HMM 14 253 S49 T63 S92 T110 N42 N47 N72 M1-T20 Signal PeptideS127 T239 N207 HMM 15 171 S43 S94 T114 M88-R112 Signal Peptide HMM 16 78S38 S43 N37 M1-G19 Signal Peptide HMM 17 71 T64 T67 M1-C19 SignalPeptide HMM 18 188 S36 T58 T133 Y31 N121 N171 M1-A21 Signal Peptide HMM19 80 S76 M1-C19 Signal Peptide HMM 20 80 M1-G25 Signal Peptide HMM 2184 S39 S53 S60 M1-G21 Signal Peptide HMM 22 171 S41 T150 M3-A21 SignalPeptide HMM 23 243 S3 S44 T75 S86 S183 N97 M1-C25 Signal Peptide S223S36 S92 S205 HMM Y40 Y110 24 311 T5 S76 T82 T93 N49 N91 N108 M1-A32Signal Peptide T109 S121 T137 N128 N135 HMM T170 S184 S11 T53 N190 S75S84 T132 S223 S274 Y69 25 57 M1-L29 Signal Peptide HMM 26 82 S46 Y26M1-S18 Signal Peptide HMM 27 115 M1-G34 Signal Peptide HMM 28 327 S93S50 S167 S233 N138 N206 M1-E25 Signal Peptide S89 T105 T214 S302 HMMT318 29 133 S63 N105 M1-E29 Signal Peptide HMM 30 129 S21 S65 T93 M1-G20Signal Peptide HMM 31 472 S164 T32 S42 T141 N61 N179 N353 M1-G20hematopoietic lineage switch 2 Signal Peptide T154 S155 T235 N356 N396(g3169729) HMM T262 T271 T334 BLAST - T376 S402 S421 GenBank S435 T441S19 S29 T327 S378 32 93 T21 M1-A18 Signal Peptide HMM 33 92 S57 S5M1-G47 SPScan 34 143 T6 T14 T135 M9-G40 Signal Peptide HMM 35 89 T15 S58S66 M1-A19 Signal Peptide HMM 36 560 T7 T76 S150 T224 N163 N184 M1-E34SPScan S228 S257 S358 N379 S474 S529 S539 T186 S219 S368 Y523 37 197 T80S163 M1-G28 Signal Peptide HMM 38 437 T47 T146 S233 S391 N46 N189 N382M1-A21 Signal Peptide S403 T43 S130 S273 HMM S339 S364 39 330 S197 T49T150 S193 N46 N64 N166 M1-G28 Signal Peptide T214 T215 T49 S111 N191 HMMS237 40 148 T73 S141 N29 N58 N71 M1-R24 receptor-activity-modifyingprotein Signal Peptide N103 (RAMP; g4165368) HMM BLAST - GenBank 41 188S49 M1-V25 Signal Peptide HMM 42 222 S89 S165 T174 T182 M1-S24 SignalPeptide T83 S155 HMM 43 111 S54 S29 S98 S50 S57 M1-T23 Signal PeptideT104 HMM 44 341 T29 S106 T120 S161 M1-G22 Signal Peptide S195 S37 S47T51 HMM S136 S223 S230 S281 45 148 S21 T63 T63 A146 N40 M1-G23 SignalPeptide HMM 46 87 S65 M1-P18 Signal Peptide HMM 47 383 T77 S95 S108 S280N93 N207 M1-P23 Signal Peptide S351 S121 S124 HMM S153 T187 48 109 S25S22 M1-L18 Signal Peptide HMM 49 185 S62 M1-A20 Signal Peptide HMM 50110 T100 T73 S97 Y48 N71 M1-C21 Signal Peptide HMM 51 126 S17 S110M1-G18 Signal Peptide HMM 52 488 S205 T31 S86 T236 N250 N321 M1-L25putative involvement in cell wall Signal Peptide S7 T447 N463 structureor biosynthesis (g3738170) HMM BLAST - GenBank 53 197 T55 S34 S46 S69T98 M1-A26 Signal Peptide S108 T119 T167 HMM S194 S2 S34 T153 54 84 S65S36 T41 S51 S69 N39 M1-G25 Signal Peptide S81 HMM 55 97 S56 M1-A22Signal Peptide HMM 56 140 S29 M1-P23 Signal Peptide HMM 57 285 S53 S108T216 S253 N153 M1-A25 Signal Peptide S277 HMM 58 262 S62 T166 S62 S71N190 M1-G28 3-acylating enzyme (Q44449) Signal Peptide Y246 HMM BLAST -GENESEQ 59 189 S120 T154 T34 T37 M1-C22 Signal Peptide S174 HMM 60 257S98 T136 T67 S112 M1-E84β SPScan S234 S237 61 82 T68 N67 M1-G18 SignalPeptide HMM 62 202 T21 S117 S120 M1-G27 Signal Peptide HMM 63 450 S107S97 S146 S339 M1-G18 Signal Peptide S440 S245 T303 HMM S304 S399 64 322T145 T214 T16 S24 N53 N130 N289 M1-G23 Signal Peptide S35 S45 T145 T269HMM S297 T300 T314 Y87 65 104 S38 S25 S75 M1-A18 Signal Peptide HMM 6693 M1 through about S18 SPscan Transmembrane: HMM M1 through about Y1767 71 S23 S64 M1 through about A24 SPscan HMM MOTIFS 68 394 S392 S393S31 S127 N53 M1 through about S31 SPScan S179 S334 T338 Transmembrane:HMM S358 T383 Y323 about M159 through about F178 MOTIFS about F109through about S127 about F225 through about V243 69 72 S59 N69 M1through about S23 SPscan Transmembrane: HMM M1 through about L16 MOTIFS70 71 S11 T26 M1 through about Q18 SPscan HMM MOTIFS 71 247 S41 T79 M1through about S25 SPscan HMM MOTIFS 72 73 S56 M1 through about G27SPscan HMM MOTIFS 73 70 M1 through about G20 SPscan HMM 74 67 M1 throughabout G30 SPscan HMM 75 91 M1 through about G26 SPScan 76 56 T29 S46 T51M1 through about S19 SPscan HMM MOTIFS 77 112 S62 S65 M1 through aboutG27 SPscan Transmembrane: HMM about W79 through about H97 MOTIFS 78 54N48 M1 through about N34 SPscan HMM MOTIFS 79 57 T33 R55 M1 throughabout C18 SPscan HMM MOTIFS 80 52 S34 M1 through about S30 SPscan HMMMOTIFS 81 64 T43 Y27 M1 through about S41 SPscan HMM MOTIFS 82 65 S45 M1through about A31 SPscan Transmembrane: HMM about L38 through about F55MOTIFS 83 56 M1 through about E23 SPscan HMM 84 120 S69 S109 N89 N95 M1through about A38 SPscan Transmembrane: about L23 HMM through about T41MOTIFS 85 67 S28 M1 through about K30 SPscan Microbodies C-terminal HMMtargetting signal: MOTIFS A65KV 86 62 S29 S42 S46 N40 M1 through aboutS29 SPscan HMM MOTIFS 87 75 S25 S46 M1 through about L19 SPscanTransmembrane: HMM about I3 through about G20 MOTIFS 88 80 T28 M1through about A20 SPscan HMM MOTIFS 89 50 S11 M1 through about C48SPscan HMM MOTIFS 90 116 S38 M1 through about G22 SPscan HMM MOTIFS 9167 S43 M1 through about P21 SPscan HMM MOTIFS 92 538 S415 S52 T77 S97N226 M1 through about S18 SPScan T178 T228 S282 Tyrosine specificprotein BLOCKS S320 S332 S384 phosphatases signature: PRINTS T401 T424S483 about V328 through about F340 MOTIFS S207 S230 S357 T410 Y263 Y36593 58 M1 through about S25 SPscan HMM 94 119 S39 M1 through about S22SPscan Transmembrane: HMM about V3 through about S21 MOTIFS 95 128 S91M1 through about G31 SPscan Transmembrane: HMM about F108 through aboutL126 MOTIFS 96 124 T115 T43 S91 M1-S20 SPScan P116-V124 HMM (urotensinII Motifs signature) BLOCKS BLAST 97 182 S28 T70 S172 S25 M1-S23, M1-S25SPScan S32 S48 S108 S131 HMM Motifs 98 237 S55 S88 S121 S135 N45 N73N107 M1-A16, M1-S21 SPScan N118 N132 C40-C198 HMM N172 N175 (cysteinespacing Motifs N185 pattern similar BLAST to that of RoBo-1) 99 160 S36S59 T143 M1-A27 SPScan HMM Motifs 100 148 T76 S64 Y103 M1-S30, M1-G31SPScan HMM Motifs 101 170 S78 T4 T30 S130 S25 M1-A23, M1-L28 SPScan S29T122 HMM Motifs 102 150 S50 S78 S91 M1-A26, M1-S28 SPScan HMM Motifs 103142 T57 T80 M1-A25, M1-G26 SPScan HMM Motifs 104 110 T3 M1-G18, M1-T25SPScan HMM Motifs 105 120 T29 S40 S72 M1-G22, M1-A20 SPScan HMM Motifs106 135 T115 S38 T41 N32 N101 M1-G26, M1-C25 SPScan HMM Motifs 107 301S53 S217 S240 S283 M1-A22 SPScan T224 HMM Motifs 108 103 S88 T73 S84M1-P19, M1-L22 SPScan HMM Motifs 109 95 T82 S52 S77 N50 M1-T15, M1-P19SPScan HMM Motifs 110 113 T84 S4 M1-P19, M1-A24 SPScan HMM Motifs 111234 S179 S184 S51 T70 N146 N191 M1-A20 NK cell activating receptorSPScan T158 S168 T228 Y29 N194 (g4493702) HMM Motifs BLAST - GenBank 112119 S39 T61 M1-G30, M1-G27 SPScan HMM Motifs 113 200 S51 T46 S191 M1-G26Signal Peptide Signal Peptide Containing Protein, SPScan Homology withKIAA0206 Motifs BLAST 114 225 M1-Q29 Signal Peptide Signal PeptideContaining Protein SPScan 115 155 S29 M1-A20 Signal Peptide SignalPeptide Containing Protein HMM Motifs 116 468 S143 T156 T227 N280 N384M1-G23 Signal Peptide Signal Peptide Containing Protein SPScan S235 T271T293 Motifs T436 S453 S117 T148 T213 S263 S417 Y73 117 403 S19 S320 S69S151 N87 M1-A24 Signal Peptide Signal Peptide Containing Protein HMMT171 T97 S393 Y193 Motifs Y378 118 131 T131 S24 T79 T118 N116 M1-G25Signal Peptide Signal Peptide Containing Protein SPScan T123 T127 Motifs119 556 T176 S192 S196 N62 N79 N127 M1-P21 Signal Peptide Signal PeptideContaining Protein, SPScan T220 S344 S369 N157 N160 L226-W244,Y402-W422, Weakly similar to Putative Motifs S476 T501 S529 V375-L392and Y355-I376 Transmembrane Protein (PTM1) HMM S541 T548 T553 S48Transmembrane Domains Precursor BLAST S115 S121 T386 T424 S500 Y104 120514 T457 T80 S86 T141 N100 N168 M1-G24 Signal Peptide Signal PeptideContaining Protein, SPScan T372 T420 S447 S94 N319 Motifs T102 S112 T240S297 S353 S470 121 109 T46 S78 T12 M1-S15 Signal Peptide Signal PeptideContaining Protein SPScan MotifS 122 431 S57 T320 S339 S396 M1-L25Signal Peptide Signal Peptide Containing Protein, SPScan S100 S239Weakly similar to OXA1L Motifs BLAST 123 142 M1-W16 Signal PeptideSignal Peptide Containing Protein SPScan 124 643 T8 S28 S77 T169 N251M1-S28 Signal Peptide, Signal Peptide Containing Protein, SPScan T199T235 S252 D37-C81, W380-C437, Thrombospondin Type 1 Protein Motifs T320S402 T413 W440-C492 and F526-C583 Pfam S414 S558 S22 T25 ThrombospondinType 1 BLAST S56 S62 S120 T184 Domains S329 T423 S475 S574 Y226 125 568S510 T24 T80 S91 N322 M1-T19 Signal Peptide Signal Peptide ContainingProtein SPScan T153 T165 S232 Motifs S248 S262 T300 T334 S380 S446 S16T19 T60 S127 S273 T436 T531 S554 T564 Y135 Y489 126 125 T62 S27 T36M1-R32 Signal Peptide, Signal Peptide Containing Protein, SPScan V4-L53Glycosyl Hydrolase Glycosyl Hydrolase Protein Motifs Family 9 ActiveSite Signature PROFILE- SCAN 127 196 T105 T47 T56 S158 M1-S26 SignalPeptide, Signal Peptide Containing Protein, SPScan H79-H123 RibosomalProtein Ribosomal Protein S18 Motifs S18 Signature BLAST Pfam PROFILE-SCAN 128 214 S112 S131 N37 N92 M1-S35 Signal Peptide Signal PeptideContaining Protein, SPScan Homology with GTP Binding Protein MotifsBLAST 129 88 M1-S24 Signal Peptide Signal Peptide Containing Protein HMM130 260 S146 S179 S192 N50 N109 M1-A48 Signal Peptide, Signal PeptideContaining Protein, SPScan S239 S70 T126 T150 G59-S142 ImmunoglobulinImmunoglobulin Superfamily Protein Motifs Domain Pfam 131 295 T176 T56S72 S179 M1-A30 Signal Peptide Signal Peptide Containing Protein SPScanS256 S87 Motifs 132 183 S11 T41 T42 S83 M1-W24 Signal Peptide, SignalPeptide Containing Protein, HMM E131-K168 and C105-H115 AdrenodoxinFamily Iron-Sulfur Motifs Adrenodoxin Iron-Sulfur Binding Protein, andBLOCKS Binding Signature, Cytochrome C Family Heme Binding PRINTSC111-V116 Cytochrome C Protein Pfam Heme Binding Signature, N69-A162Iron-Sulfur Cluster Binding Domain 133 113 S93 T89 Y9 M1-G30 SignalPeptide, Signal Peptide Containing Protein, SPScan V28-L74 PF00646 F-BoxPF00646 F-Box Protein Motifs Domain Pfam 134 160 T46 T55 S65 S124 M1-A27Signal Peptide Signal Peptide Containing Protein, SPScan T125 T46F45G2.10 and Yhr122wp Homology Motifs BLAST

TABLE 3 Nucleotide Disease/Condition-Specific Expression (Total of SEQID NO: Tissue Expression (Fraction of Total) Fraction) Vector 135Hematopoietic/Immune (1.000) Inflammation (1.000) pBLUESCRIPT 136Hematopoietic/Immune (0.750) Cardiovascular (0.250) Inflammation (0.750)Cancer (0.250) pSPORT1 137 Nervous (1.000) Trauma (1.000) pSPORT1 138Musculoskeletal (1.000) Inflammation (1.000) pSPORT1 139Gastrointestinal (0.714) Cardiovascular (0.143) Cancer (0.714) Trauma(0.143) pSPORT1 Reproductive (0.143) 140 Nervous (1.000) Neurological(0.500) Trauma (0.500) pSPORT1 141 Reproductive (0.293) Gastrointestinal(0.146) Cancer (0.524) Inflammation (0.256) Fetal (0.146) pSPORT1Hematopoietic/Immune (0.146) 142 Reproductive (0.266) Gastrointestinal(0.170) Cancer (0.479) Inflammation (0.277) Fetal (0.181) pINCY Nervous(0.138) 143 Reproductive (0.417) Nervous (0.292) Cancer (0.417)Inflammation (0.250) Fetal (0.167) pINCY Developmental (0.125) 144Reproductive (0.321) Cardiovascular (0.143) Cancer (0.464) Fetal (0.214)Inflammation (0.143) pINCY Developmental (0.143) 145 Reproductive(0.600) Gastrointestinal (0.400) Cancer (0.400) Trauma (0.400)Inflammation (0.200) pINCY 146 Cardiovascular (0.400) Dermatologic(0.200) Cancer (0.600) Fetal (0.600) pINCY Nervous (0.200) 147Developmental (0.667) Gastrointestinal (0.333) Fetal (0.667) Cancer(0.333) pINCY 148 Reproductive (0.256) Nervous (0.248) Cancer (0.479)Inflammation (0.214) Fetal (0.145) pINCY Cardiovascular (0.137) 149Reproductive (0.244) Nervous (0.178) Cancer (0.433) Inflammation (0.322)Fetal (0.156) pINCY Hematopoietic/Immune (0.167) 150 Cardiovascular(0.923) Developmental (0.077) Cancer (0.692) Fetal (0.154) Inflammation(0.154) pINCY 151 Reproductive (0.215) Nervous (0.190) Cancer (0.494)Inflammation (0.278) Trauma (0.152) pINCY Gastrointestinal (0.177) 152Reproductive (0.200) Nervous (0.171) Inflammation (0.371) Cancer (0.229)Fetal (0.200) pINCY Hematopoietic/Immune (0.143) 153 Reproductive(0.333) Nervous (0.157) Cancer (0.549) Inflammation (0.176) Fetal(0.137) pINCY Hematopoietic/Immune (0.137) 154 Gastrointestinal (0.500)Urologic (0.167) Inflammation (0.667) Cancer (0.167) Trauma (0.167)pINCY 155 Gastrointestinal (0.429) Reproductive (0.286) Inflammation(0.429) Cancer (0.286) Trauma (0.143) pINCY Nervous (0.143) 156Reproductive (1.000) Cancer (0.500) Inflammation (0.500) pINCY 157Hematopoietic/Immune (0.346) Reproductive (0.154) Cancer (0.404)Inflammation (0.404) Fetal (0.212) pINCY Gastrointestinal (0.115) 158Reproductive (0.236) Hematopoietic/Immune (0.217) Cancer (0.415)Inflammation (0.358) Fetal (0.142) pINCY Gastrointestinal (0.132) 159Gastrointestinal (1.000) Cancer (1.000) pSPORT1 160 Developmental(0.500) Hematopoietic/Immune (0.250) Fetal (0.500) Inflammation (0.250)Trauma (0.250) pINCY Nervous (0.250) 161 Hematopoietic/Immune (0.250)Reproductive (0.250) Cancer (0.583) Fetal (0.292) Inflammation (0.250)pINCY Nervous (0.208) 162 Gastrointestinal (0.412) Reproductive (0.412)Cancer (0.735) Inflammation (0.176) Fetal (0.029) pINCY Cardiovascular(0.088) 163 Reproductive (0.298) Cardiovascular (0.170) Nervous Cancer(0.532) Inflammation (0.213) Fetal (0.191) pINCY (0.149) 164Gastrointestinal (0.333) Hematopoietic/Immune (0.333) Cancer (0.667)Inflammation (0.333) pINCY Reproductive (0.333) 165 Reproductive (0.295)Gastrointestinal (0.159) Cancer (0.534) Inflammation (0.284) Fetal(0.091) pINCY Nervous (0.148) 166 Hematopoietic/Immune (0.538)Cardiovascular (0.077) Inflammation (0.731) Cancer (0.154) Fetal (0.154)pINCY Reproductive (0.077) 167 Reproductive (0.483) Gastrointestinal(0.121) Cancer (0.672) Inflammation (0.155) pINCY Nervous (0.103) 168Gastrointestinal (0.222) Hematopoietic/Immune (0.222) Cancer (0.519)Inflammation (0.370) Fetal (0.259) pINCY Nervous (0.148) 169 Urologic(1.000) Cancer (0.333) Fetal (0.333) Inflammation (0.333) pINCY 170Reproductive (0.214) Gastrointestinal (0.179) Cancer (0.643)Inflammation (0.143) Fetal (0.107) pINCY Nervous (0.143) 171Reproductive (0.261) Developmental (0.174) Cancer (0.391) Fetal (0.304)Inflammation (0.217) pINCY Nervous (0.174) 172 Reproductive (0.357)Gastrointestinal (0.321) Cancer (0.571) Inflammation (0.286) Fetal(0.107) pINCY Cardiovascular (0.071) 173 Reproductive (0.306) Nervous(0.161) Cancer (0.387) Inflammation (0.323) Fetal (0.226) pINCYCardiovascular (0.129) 174 Reproductive (0.229) Nervous (0.188) Cancer(0.521) Inflammation (0.312) Trauma (0.146) pSPORT1 Cardiovascular(0.167) 175 Reproductive (0.444) Developmental (0.167) Cancer (0.556)Fetal (0.278) Trauma (0.111) pSPORT1 Cardiovascular (0.111) 176Reproductive (0.294) Gastrointestinal (0.176) Cancer (0.765) Fetal(0.118) Inflammation (0.118) pSPORT1 Cardiovascular (0.118) 177Gastrointestinal (1.000) Cancer (0.667) Inflammation (0.333) pINCY 178Reproductive (0.385) Nervous (0.231) Cancer (0.385) Inflammation (0.385)pINCY Gastrointestinal (0.154) 179 Reproductive (0.500) Cardiovascular(0.167) Cancer (0.667) Fetal (0.167) Inflammation (0.167) pBLUESCRIPTGastrointestinal (0.167) 180 Cardiovascular (0.231) Reproductive (0.231)Cancer (0.615) Inflammation (0.308) Fetal (0.154) pINCY Gastrointestinal(0.154) 181 Reproductive (0.324) Gastrointestinal (0.176) Cancer (0.519)Inflammation (0.222) Fetal (0.157) pINCY Cardiovascular (0.130) 182Reproductive (0.320) Nervous (0.180) Cancer (0.580) Inflammation (0.160)Fetal (0.100) pINCY Gastrointestinal (0.120) 183 Gastrointestinal(0.667) Reproductive (0.333) Cancer (1.000) pINCY 184 Urologic (0.667)Dermatologic (0.333) Cancer (0.667) Fetal (0.333) pSPORT1 185Cardiovascular (0.500) Reproductive (0.500) Cancer (1.000) pINCY 186Reproductive (0.393) Developmental (0.107) Cancer (0.607) Fetal (0.179)Inflammation (0.107) pINCY Urologic (0.107) 187 Cardiovascular (0.400)Reproductive (0.333) Inflammation (0.467) Cancer (0.267) Fetal (0.267)pSPORT1 Gastrointestinal (0.133) 188 Nervous (0.318) Reproductive(0.227) Urologic (0.136) Cancer (0.636) Inflammation (0.136) Trauma(0.091) pINCY 189 Cardiovascular (0.500) Reproductive (0.500) Cancer(1.000) pINCY 190 Reproductive (0.318) Nervous (0.227) Cancer (0.500)Fetal (0.227) Inflammation (0.227) pINCY Hematopoietic/Immune (0.136)191 Reproductive (0.253) Cardiovascular (0.158) Cancer (0.463)Inflammation (0.232) Fetal (0.200) pINCY Gastrointestinal (0.147) 192Reproductive (0.333) Gastrointestinal (0.286) Cancer (0.571)Inflammation (0.333) Fetal (0.095) pINCY Cardiovascular (0.095) 193Reproductive (0.304) Cardiovascular (0.217) Cancer (0.435) Inflammation(0.391) Fetal (0.174) pINCY Gastrointestinal (0.130) 194 Reproductive(0.312) Nervous (0.188) Cancer (0.438) Inflammation (0.250) Fetal(0.188) pINCY Cardiovascular (0.125) 195 Developmental (1.000) Fetal(1.000) pINCY 196 Reproductive (0.233) Cardiovascular (0.209) Cancer(0.605) Fetal (0.186) Inflammation (0.116) pINCY Nervous (0.140) 197Reproductive (0.182) Gastrointestinal (0.136) Cancer (0.477)Inflammation (0.341) Fetal (0.182) pINCY Hematopoietic/Immune (0.136)198 Gastrointestinal (0.205) Reproductive (0.205) Inflammation (0.341)Cancer (0.250) Fetal (0.227) pINCY Cardiovascular (0.114) 199Cardiovascular (0.520) Reproductive (0.280) Cancer (0.720) Fetal (0.200)Inflammation (0.080) pINCY Developmental (0.160) 200 Lung (0.958)Developmental (0.25) Cancer (0.583) Fetal or Proliferating (0.292)pBLUESCRIPT Musculoskeletal (0.042) Inflammation (0.167) 201Reproductive (0.571) Musculoskeletal (0.143) Cancer (0.429) Inflammation(0.571) pSPORT1 Nervous (0.143) Urologic (0.143) 202 Endocrine (0.250)Nervous (0.250) Cardiovascular (0.125) Cancer (0.375) Inflammation(0.625) pSPORT1 Developmental (0.125) Gastrointestinal (0.125) Fetal orProliferating (0.125) Reproductive (0.125) 203 Lung (1.000) Fetal orProliferating (1.000) pINCY 204 Lung (0.500) Penis (0.500) Cancer(0.500) pINCY 205 Cardiovascular (0.231) Dermatologic (0.231) Fetal orProliferating (0.385) Cancer (0.308) pINCY Reproductive (0.231) 206Nervous (0.596) Reproductive (0.154) Cancer (0.442) Neurological (0.192)pINCY Gastrointestinal (0.077) Inflammation (0.231) 207 Gastrointestinal(1.000) Inflammation (1.000) pINCY 208 Reproductive (0.300)Hematopoietic/Immune (0.200) Cancer (0.450) Inflammation (0.400) pSPORT1Nervous (0.150) Fetal or Proliferating (0.250) 209 Heart (0.500) Brain(0.500) Neurological (0.500) Inflammation (0.500) pINCY 210 Nervous(0.625) Reproductive (0.250) Cancer (0.750) Fetal or Proliferating(0.250) pINCY Musculoskeletal (0.125) Neurological (0.125) 211 Nervous(0.261) Reproductive (0.304) Cancer (0.522) Fetal or Proliferating(0.174) pSPORT1 Gastrointestinal (0.174) Inflammation (0.130) 212 Testis(1.000) Inflammation (1.000) pBLUESCRIPT 213 Nervous (0.400)Reproductive (0.400) Cancer (0.400) Inflammation (0.400) pBLUESCRIPTGastrointestinal (0.200) Neurological (0.200) 214 Reproductive (0.476)Gastrointestinal (0.286) Cancer (0.714) Inflammation (0.286) pSPORT1Cardiovascular (0.095) Neurological (0.048) 215 Reproductive (0.284)Gastrointestinal (0.216) Cancer (0.486) Inflammation (0.351) pSPORT1Nervous (0.176) Hematopoietic/Immune (0.108) Fetal or Proliferating(0.122) Cardiovascular (0.108) 216 Uterus (0.500) Prostate (0.500)Cancer (0.500) Inflammation (0.500) pINCY 217 Nervous (0.429)Cardiovascular (0.143) Cancer (0.571) Inflammation (0.429) pSPORT1Gastrointestinal (0.143) Hematopoietic/Immune (0.143) Fetal orProliferating (0.285) Reproductive (0.143) 218 Reproductive (0.450)Hematopoietic/Immune (0.200) Cancer (0.650) Inflammation (0.200) pINCYNervous (0.100) Gastrointestinal (0.100) Fetal or Proliferating (0.050)219 Reproductive (0.364) Cardiovascular (0.182) Cancer (0.636) Fetal orProliferating (0.182) pINCY Nervous (0.182) Inflammation (0.273) 220Prostate (1.000) Inflammation (1.000) pSPORT1 221 Developmental (0.333)Nervous (0.333) Cancer (0.667) Fetal or Proliferating (0.667) pSPORT1Reproductive (0.333) 222 Reproductive (0.393) Hematopoietic/Immune(0.180) Cancer (0.508) Inflammation (0.344) pSPORT1 Nervous (0.098)Cardiovascular (0.098) Fetal or Proliferating (0.066) 223 Endocrine(0.333) Gastrointestinal (0.333) Cancer (1.000) pINCY Reproductive(0.333) 224 Cardiovascular (0.200) Developmental (0.200) Cancer (0.800)Fetal or Proliferating (0.200) pINCY Gastrointestinal (0.200)Reproductive (0.200) Urologic (0.200) 225 Lung (1.000) Cancer (1.000)pINCY 226 Reproductive (0.302) Hematopoietic/Immune (0.254) Cancer(0.381) Inflammation (0.381) pSPORT1 Cardiovascular (0.111) Fetal orProliferating (0.286) 227 Lymphocytes (1.000) Inflammation (1.000) pINCY228 Cardiovascular (0.531) Reproductive (0.250) Cancer (0.656)Inflammation (0.250) pINCY Urologic (0.094) Fetal or Proliferating(0.094) 229 Reproductive (0.333) Cardiovascular (0.167) Cancer (0.500)Fetal or Proliferating (0.167) pINCY Gastrointestinal (0.167) Endocrine(0.167) Inflammation (0.333) Hematopoietic/Immune (0.167) 230Hematopoietic/Immune (0.500) Reproductive (0.500) Cell Proliferation(0.500) pBLUESCRIPT Inflammation (0.500) 231 Cardiovascular (0.333)Nervous (0.333) 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TABLE 4 Polynucleotide SEQ ID NO: Clone ID Library Library Description135 443531 MPHGNOT03 The library was constructed using RNA isolated fromplastic adherent mononuclear cells isolated from buffy coat unitsobtained from unrelated male and female donors. 136 632860 NEUTGMT01 Thelibrary was constructed using RNA isolated from peripheral bloodgranulocytes collected by density gradient centrifugation throughFicoll-Hypaque. The cells were isolated from buffy coat units obtainedfrom 20 unrelated male and female donors. Cells were cultured in 10 nMGM-CSF for 1 hour before washing and harvesting for RNA preparation. 137670010 CRBLNOT01 The library was constructed using RNA isolated from thecerebellum tissue of a 69-year-old Caucasian male who died from chronicobstructive pulmonary disease. Patient history included myocardialinfarction, hypertension, and osteoarthritis. 138 726498 SYNOOAT01 Thelibrary was constructed using RNA isolated from the knee synovialmembrane tissue of an 82- year-old female with osteoarthritis. 139795064 OVARNOT03 The library was constructed using RNA isolated fromovarian tissue removed from a 43-year-old Caucasian female duringremoval of the fallopian tubes and ovaries. Pathology for the associatedtumor tissue indicated grade 2 mucinous cystadenocarcinoma. Patienthistory included mitral valve disorder, pneumonia, and viral hepatitis.Family history included atherosclerotic coronary artery disease,pancreatic cancer, cerebrovascular disease, breast cancer, and uterinecancer. 140 924925 BRAINOT04 The library was constructed using RNAisolated from the brain tissue of a 44-year-old Caucasian male with acerebral hemorrhage. The tissue, which contained coagulated blood, camefrom the choroid plexus of the right anterior temporal lobe. Familyhistory included coronary artery disease and myocardial infarction. 141962390 BRSTTUT03 The library was constructed using RNA isolated frombreast tumor tissue removed from a 58-year- old Caucasian female duringa unilateral extended simple mastectomy. Pathology indicatedmulticentric invasive grade 4 lobular carcinoma. The mass was identifiedin the upper outer quadrant, and three separate nodules were found inthe lower outer quadrant of the left breast. Patient history includedskin cancer, rheumatic heart disease, osteoarthritis, and tuberculosis.Family history included cerebrovascular disease, coronary arteryaneurysm, breast cancer, prostate cancer, atherosclerotic coronaryartery disease, and type I diabetes. 142 1259405 MENITUT03 The librarywas constructed using RNA isolated from brain meningioma tissue removedfrom a 35- year-old Caucasian female during excision of a cerebralmeningeal lesion. Pathology indicated a benign neoplasm in the rightcerebellopontine angle of the brain. Patient history includedhypothyroidism. Family history included myocardial infarction and breastcancer. 143 1297384 BRSTNOT07 The library was constructed using RNAisolated from diseased breast tissue removed from a 43- year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology indicated mildly proliferative fibrocystic changes withepithelial hyperplasia, papillomatosis, and duct ectasia. Pathology forthe associated tumor tissue indicated invasive grade 4, nuclear grade 3mammary adenocarcinoma with extensive comedo necrosis. Family historyincluded epilepsy, atherosclerotic coronary artery disease, and type IIdiabetes. 144 1299627 BRSTNOT07 The library was constructed using RNAisolated from diseased breast tissue removed from a 43- year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology indicated mildly proliferative fibrocystic changes withepithelial hyperplasia, papillomatosis, and duct ectasia. Pathology forthe associated tumor tissue indicated invasive grade 4, nuclear grade 3mammary adenocarcinoma with extensive comedo necrosis. Family historyincluded epilepsy, atherosclerotic coronary artery disease, and type IIdiabetes. 145 1306026 PLACNOT02 The library was constructed using RNAisolated from the placental tissue of a Hispanic female fetus, who wasprematurely delivered at 21 weeks' gestation. Serologies of the mother'sblood were positive for CMV (cytomegalovirus). 146 1316219 BLADTUT02 Thelibrary was constructed using RNA isolated from bladder tumor tissueremoved from an 80- year-old Caucasian female during a radicalcystectomy and lymph node excision. Pathology indicated grade 3 invasivetransitional cell carcinoma. Family history included osteoarthritis andatherosclerosis. 147 1329031 PANCNOT07 The library was constructed usingRNA isolated from the pancreatic tissue of a Caucasian male fetus, whodied at 23 weeks' gestation. 148 1483050 CORPNOT02 The library wasconstructed using RNA isolated from diseased corpus callosum tissueremoved from the brain of a 74-year-old Caucasian male who died fromAlzheimer's disease. 149 1514160 PANCTUT01 The library was constructedusing RNA isolated from pancreatic tumor tissue removed from a 65-year-old Caucasian female during radical subtotal pancreatectomy.Pathology indicated an invasive grade 2 adenocarcinoma. Patient historyincluded type II diabetes, osteoarthritis, cardiovascular disease,benign neoplasm in the large bowel, and a cataract. Family historyincluded cardiovascular disease, type II diabetes, and stomach cancer.150 1603403 LUNGNOT15 The library was constructed using RNA isolatedfrom lung tissue removed from a 69-year-old Caucasian male during asegmental lung resection. Pathology for the associated tumor tissueindicated residual grade 3 invasive squamous cell carcinoma. Patienthistory included acute myocardial infarction, prostatic hyperplasia, andmalignant skin neoplasm. Family history included cerebrovasculardisease, type I diabetes, acute myocardial infarction, andarteriosclerotic coronary disease. 151 1652303 PROSTUT08 The library wasconstructed using RNA isolated from prostate tumor tissue removed from a60- year-old Caucasian male during radical prostatectomy and regionallymph node excision. Pathology indicated an adenocarcinoma (Gleasongrade 3 + 4). Adenofibromatous hyperplasia was also present. The patientpresented with elevated prostate specific antigen (PSA). Patient historyincluded a kidney cyst. Family history included tuberculosis,cerebrovascular disease, and arteriosclerotic coronary artery disease.152 1693358 COLNNOT23 The library was constructed using RNA isolatedfrom diseased colon tissue removed from a 16- year-old Caucasian maleduring a total colectomy with abdominal/perineal resection. Pathologyindicated gastritis and pancolonitis consistent with the acute phase ofulcerative colitis. There was only mild involvement of the ascending andsigmoid colon, and no significant involvement of the cecum, rectum, orterminal ileum. Family history included irritable bowel syndrome. 1531707711 DUODNOT02 The library was constructed using RNA isolated fromduodenal tissue of a 8-year-old Caucasian female, who died from headtrauma. Serology was positive for cytomegalovirus (CMV). 154 1738735COLNNOT22 The library was constructed using RNA isolated from colontissue removed from a 56-year-old Caucasian female with Crohn's diseaseduring a partial resection of the small intestine. Pathology indicatedCrohn's disease of the ileum and ileal-colonic anastomosis, causing afistula at the anastomotic site that extended into pericolonic fat. Theileal mucosa showed linear and puncture ulcers with intervening normaltissue. Previous surgeries included a partial ileal resection andpermanent ileostomy. Family history included irritable bowel syndrome.155 1749147 STOMTUT02 The library was constructed using RNA isolatedfrom stomach tumor tissue obtained from a 68- year-old Caucasian femaleduring a partial gastrectomy. Pathology indicated a malignant lymphomaof diffuse large-cell type. Patient history included thalassemia. Familyhistory included acute leukemia, malignant neoplasm of the esophagus,malignant stomach neoplasm, and atherosclerotic coronary artery disease.156 1817722 PROSNOT20 The library was constructed using RNA isolatedfrom diseased prostate tissue removed from a 65- year-old Caucasian maleduring a radical prostatectomy. Pathology indicated adenofibromatoushyperplasia. Pathology for the associated tumor tissue indicated anadenocarcinoma. 157 1831290 THP1AZT01 The library was constructed using1 microgram of polyA RNA isolated from THP-1 promonocyte cells treatedfor three days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCCTIB 202) is a human promonocyte line derived from peripheral blood of a1-year-old Caucasian male with acute monocytic leukemia. 158 1831477THP1AZT01 The library was constructed using 1 microgram of polyA RNAisolated from THP-1 promonocyte cells treated for three days with 0.8micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is a humanpromonocyte line derived from peripheral blood of a 1-year-old Caucasianmale with acute monocytic leukemia. 159 1841607 COLNNOT07 The librarywas constructed using RNA isolated from colon tissue removed from a60-year-old Caucasian male during a left hemicolectomy. 160 1852391LUNGFET03 The library was constructed using RNA isolated from lungtissue removed from a Caucasian female fetus, who died at 20 weeks'gestation. 161 1854555 HNT3AZT01 Library was constructed using RNAisolated from the hNT2 cell line (derived from a human teratocarcinomathat exhibited properties characteristic of a committed neuronalprecursor). Cells were treated for three days with 0.35 micromolar5-aza-2′-deoxycytidine (AZT). 162 1855755 PROSNOT18 The library wasconstructed using RNA isolated from diseased prostate tissue removedfrom a 58- year-old Caucasian male during a radical cystectomy, radicalprostatectomy, and gastrostomy. Pathology indicated adenofibromatoushyperplasia. This tissue was associated with a grade 3 transitional cellcarcinoma. Patient history included angina and emphysema. Family historyincluded acute myocardial infarction, atherosclerotic coronary arterydisease, and type II diabetes. 163 1861434 PROSNOT19 The library wasconstructed using RNA isolated from diseased prostate tissue removedfrom a 59- year-old Caucasian male during a radical prostatectomy withregional lymph node excision. Pathology indicated adenofibromatoushyperplasia. Pathology for the associated tumor tissue indicated anadenocarcinoma (Gleason grade 3 + 3). The patient presented withelevated prostate- specific antigen (PSA). Patient history includedcolon diverticuli and thrombophlebitis. Family history included benignhypertension, multiple myeloma, hyperlipidernia and rheumatoidarthritis. 164 1872334 LEUKNOT02 The library was constructed using RNAisolated from white blood cells of a 45-year-old female with blood typeO+. The donor tested positive for cytomegalovirus (CMV). 165 1877230LEUKNOT03 The library was constructed using RNA isolated from whiteblood cells of a 27-year-old female with blood type A+. The donor testednegative for cytomegalovirus (CMV). 166 1877885 LEUKNOT03 The librarywas constructed using RNA isolated from white blood cells of a27-year-old female with blood type A+. The donor tested negative forcytomegalovirus (CMV). 167 1889269 BLADTUT07 The library was constructedusing RNA isolated from bladder tumor tissue removed from the anteriorbladder wall of a 58-year-old Caucasian male during a radicalcystectomy, radical prostatectomy, and gastrostomy. Pathology indicateda grade 3 transitional cell carcinoma in the left lateral bladder.Patient history included angina and emphysema. Family history includedacute myocardial infarction, atherosclerotic coronary artery disease,and type II diabetes. 168 1890243 BLADTUT07 The library was constructedusing RNA isolated from bladder tumor tissue removed from the anteriorbladder wall of a 58-year-old Caucasian male during a radicalcystectomy, radical prostatectomy, and gastrostomy. Pathology indicateda grade 3 transitional cell carcinoma in the left lateral bladder.Patient history included angina and emphysema. Family history includedacute myocardial infarction, atherosclerotic coronary artery disease,and type II diabetes. 169 1900433 BLADTUT06 The library was constructedusing RNA isolated from bladder tumor tissue removed from the posteriorbladder wall of a 58-year-old Caucasian male during a radicalcystectomy, radical prostatectomy, and gastrostomy. Pathology indicatedgrade 3 transitional cell carcinoma in the left lateral bladder wall.Patient history included angina and emphysema. Family history includedacute myocardial infarction, atherosclerotic coronary artery disease,and type II diabetes. 170 1909441 CONNTUT01 The library was constructedusing RNA isolated from a soft tissue tumor removed from the clival areaof the skull of a 30-year-old Caucasian female. Pathology indicatedchondroid chordoma with neoplastic cells reactive for keratin. 1711932226 COLNNOT16 The library was constructed using RNA isolated fromsigmoid colon tissue removed from a 62- year-old Caucasian male during asigmoidectomy and permanent colostomy. 172 1932647 COLNNOT16 The librarywas constructed using RNA isolated from sigmoid colon tissue removedfrom a 62- year-old Caucasian male during a sigmoidectomy and permanentcolostomy. 173 2124245 BRSTNOT07 The library was constructed using RNAisolated from diseased breast tissue removed from a 43- year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology indicated mildly proliferative fibrocystic changes withepithelial hyperplasia, papillomatosis, and duct ectasia. Pathology forthe associated tumor tissue indicated invasive grade 4, nuclear grade 3mammary adenocarcinoma with extensive comedo necrosis. Family historyincluded epilepsy, atherosclerotic coronary artery disease, and type IIdiabetes. 174 2132626 OVARNOT03 The library was constructed using RNAisolated from ovarian tissue removed from a 43-year-old Caucasian femaleduring removal of the fallopian tubes and ovaries. Pathology for theassociated tumor tissue indicated grade 2 mucinous cystadenocarcinoma.Patient history included mitral valve disorder, pneumonia, and viralhepatitis. Family history included atherosclerotic coronary arterydisease, pancreatic cancer, cerebrovascular disease, breast cancer, anduterine cancer. 175 2280639 PROSNON01 The library was constructed andnormalized from 4.4 million independent clones from the PROSNOT11library. Starting RNA was made from prostate tissue removed from a28-year-old Caucasian male who died from a gunshot wound. Thenormalization and hybridization conditions were adapted from Soares, M.B. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 9228-9232, using alonger (19 hour) reannealing hybridization period. 176 2292356 BRAINON01The library was constructed and normalized from 4.88 million independentclones from the BRAINOT03 library. Starting RNA was made from braintissue removed from a 26-year-old Caucasian male during cranioplasty andexcision of a cerebral meningeal lesion. Pathology for the associatedtumor tissue indicated a grade 4 oligoastrocytoma in the rightfronto-parietal part of the brain. 177 2349310 COLSUCT01 The library wasconstructed using RNA isolated from diseased sigmoid colon tissueobtained from a 70-year-old Caucasian male during colectomy withpermanent ileostomy. Pathology indicated chronic ulcerative colitis.Patient history included benign neoplasm of the colon. Family historyincluded atherosclerotic coronary artery disease and myocardialinfarctions. 178 2373227 ADRENOT07 The library was constructed using RNAisolated from adrenal tissue removed from a 61-year-old female during abilateral adrenalectomy. Patient history included an unspecifieddisorder of the adrenal glands. 179 2457682 ENDANOT01 The library wasconstructed using RNA isolated from aortic endothelial cell tissue froman explanted heart removed from a male during a heart transplant. 1802480426 SMCANOT01 The library was constructed using RNA isolated from anaortic smooth muscle cell line derived from the explanted heart of amale during a heart transplant. 181 2503743 CONUTUT01 The library wasconstructed using RNA isolated from sigmoid mesentery tumor tissueobtained from a 61-year-old female during a total abdominal hysterectomyand bilateral salpingo- oophorectomy with regional lymph node excision.Pathology indicated a metastatic grade 4 malignant mixed mullerian tumorpresent in the sigmoid mesentery at two sites. 182 2537684 BONRTUT01 Thelibrary was constructed using RNA isolated from rib tumor tissue removedfrom a 16-year-old Caucasian male during a rib osteotomy and a wedgeresection of the lung. Pathology indicated a metastatic grade 3 (of 4)osteosarcoma, forming a mass involving the chest wall. 183 2593853OVARTUT02 The library was constructed using RNA isolated from ovariantumor tissue removed from a 51- year-old Caucasian female during anexploratory laparotomy, total abdominal hysterectomy,salpingo-oophorectomy, and an incidental appendectomy. Pathologyindicated mucinous cystadenoma presenting as a multiloculated neoplasminvolving the entire left ovary. The right ovary contained a follicularcyst and a hemorrhagic corpus luteum. The uterus showed proliferativeendometrium and a single intramural leiomyoma. The peritoneal biopsyindicated benign glandular inclusions consistent with endosalpingiosis.Family history included atherosclerotic coronary artery disease, benignhypertension, breast cancer, and uterine cancer. 184 2622354 KERANOT02The library was constructed using RNA isolated from epidermal breastkeratinocytes (NHEK). NHEK (Clontech #CC-2501) is a human breastkeratinocyte cell line derived from a 30-year-old black female duringbreast-reduction surgery. 185 2641377 LUNGTUT08 The library wasconstructed using RNA isolated from lung tumor tissue removed from a63-year- old Caucasian male during a right upper lobectomy withfiberoptic bronchoscopy. Pathology indicated a grade 3 adenocarcinoma.Patient history included atherosclerotic coronary artery disease, anacute myocardial infarction, rectal cancer, an asymptomatic abdominalaortic aneurysm, and cardiac dysrhythmia. Family history includedcongestive heart failure, stomach cancer, and lung cancer, type IIdiabetes, atherosclerotic coronary artery disease, and an acutemyocardial infarction. 186 2674857 KIDNNOT19 The library was constructedusing RNA isolated from kidney tissue removed a 65-year-old Caucasianmale during an exploratory laparotomy and nephroureterectomy. Pathologyfor the associated tumor tissue indicated a grade 1 renal cell carcinomawithin the upper pole of the left kidney. Patient history includedmalignant melanoma of the abdominal skin, benign neoplasm of colon,cerebrovascular disease, and umbilical hernia. Family history includedmyocardial infarction, atherosclerotic coronary artery disease,cerebrovascular disease, prostate cancer, myocardial infarction, andatherosclerotic coronary artery disease. 187 2758485 THP1AZS08 Thesubtracted THP-1 promonocyte cell line library was constructed using5.76 million clones from a 5-aza-2′-deoxycytidine (AZT) treated THP-1cell library. Starting RNA was made from THP-1 promonocyte cells treatedfor three days with 0.8 micromolar AZT. The library was oligo(dT)-primed, and cDNAs were cloned directionally into the pSPORT1 vectoringsystem using Sal1 (5′) and NotI (3′). The hybridization probe forsubtraction was derived from a similarly constructed library, made from1 microgram of polyA RNA isolated from untreated THP-1 cells. 5.76million clones from the AZ-treated THP-1 cell library were thensubjected to two rounds of subtractive hybridization with 5 millionclones from the untreated THP-1 cell library. Subtractive hybridizationconditions were based on the methodologies of Swaroop et al. (Nucl.Acids Res. (1991) 19: 1954) and Bonaldo et al. (Genome Res (1996) 6:791-806). 188 2763296 BRSTNOT12 The library was constructed using RNAisolated from diseased breast tissue removed from a 32- year-oldCaucasian female during a bilateral reduction mammoplasty. Pathologyindicated nonproliferative fibrocystic disease. Family history includedbenign hypertension and atherosclerotic coronary artery disease. 1892779436 OVARTUT03 The library was constructed using RNA isolated fromovarian tumor tissue removed from the left ovary of a 52-year-old mixedethnicity female during a total abdominal hysterectomy, bilateralsalpingo-oophorectomy, peritoneal and lymphatic structure biopsy,regional lymph node excision, and peritoneal tissue destruction.Pathology indicated an invasive grade 3 (of 4) seroanaplastic carcinomaforming a mass in the left ovary. The endometrium was atrophic. Multiple(2) leiomyomata were identified, one subserosal and 1 intramural.Pathology also indicated a metastatic grade 3 seroanaplastic carcinomainvolving the omentum, cul-de-sac peritoneum, left broad ligamentperitoneum, and mesentery colon. Patient history included breast cancer,chronic peptic ulcer, and joint pain. Family history included coloncancer, cerebrovascular disease, breast cancer, type II diabetes,esophagus cancer, and depressive disorder. 190 2808528 BLADTUT08 Thelibrary was constructed using RNA isolated from bladder tumor tissueremoved from a 72- year-old Caucasian male during a radical cystectomyand prostatectomy. Pathology indicated an invasive grade 3 (of 3)transitional cell carcinoma in the right bladder base. Family historyincluded myocardial infarction, cerebrovascular disease, brain cancer,and myocardial infarction. 191 2809230 BLADTUT08 The library wasconstructed using RNA isolated from bladder tumor tissue removed from a72- year-old Caucasian male during a radical cystectomy andprostatectomy. Pathology indicated an invasive grade 3 (of 3)transitional cell carcinoma in the right bladder base. Patient historyincluded pure hypercholesterolemia and tobacco abuse. Family historyincluded myocardial infarction, cerebrovascular disease, brain cancer,and myocardial infarction. 192 2816821 BRSTNOT14 The library wasconstructed using RNA isolated from breast tissue removed from a62-year-old Caucasian female during a unilateral extended simplemastectomy. Pathology for the associated tumor tissue indicated aninvasive grade 3 (of 4), nuclear grade 3 (of 3) adenocarcinoma, ductaltype. Ductal carcinoma in situ, comedo type, comprised 60% of the tumormass. Metastatic adenocarcinoma was identified in one (of 14) axillarylymph nodes with no perinodal extension. The tumor cells were stronglypositive for estrogen receptors and weakly positive for progesteronereceptors. Patient history included a benign colon neoplasm,hyperlipidemia, and cardiac dysrhythmia. Family history includedatherosclerotic coronary artery disease, myocardial infarction, coloncancer, ovarian cancer, lung cancer, and cerebrovascular disease. 1932817268 BRSTNOT14 The library was constructed using RNA isolated frombreast tissue removed from a 62-year-old Caucasian female during aunilateral extended simple mastectomy. Pathology for the associatedtumor tissue indicated an invasive grade 3 (of 4), nuclear grade 3 (of3) adenocarcinoma, ductal type. Ductal carcinoma in situ, comedo type,comprised 60% of the tumor mass. Metastatic adenocarcinoma wasidentified in one (of 14) axillary lymph nodes with no perinodalextension. The tumor cells were strongly positive for estrogen receptorsand weakly positive for progesterone receptors. Patient history includeda benign colon neoplasm, hyperlipidemia, and cardiac dysrhythmia. Familyhistory included atherosclerotic coronary artery disease, myocardialinfarction, colon cancer, ovarian cancer, lung cancer, andcerebrovascular disease. 194 2923165 SININOT04 The library wasconstructed using RNA isolated from diseased ileum tissue obtained froma 26- year-old Caucasian male during a partial colectomy, permanentcolostomy, and an incidental appendectomy. Pathology indicatedmoderately to severely active Crohn's disease. Family history includedenteritis of the small intestine. 195 2949822 KIDNFET01 The library wasconstructed using RNA isolated from kidney tissue removed from aCaucasian female fetus, who died at 17 weeks' gestation fromanencephalus. 196 2992192 KIDNFET02 The library was constructed usingRNA isolated from kidney tissue removed from a Caucasian male fetus, whowas stillborn with a hypoplastic left heart and died at 23 weeks'gestation. 197 2992458 KIDNFET02 The library was constructed using RNAisolated from kidney tissue removed from a Caucasian male fetus, who wasstillborn with a hypoplastic left heart and died at 23 weeks' gestation.198 3044710 HEAANOT01 The library was constructed using RNA isolatedfrom right coronary and right circumflex coronary artery tissue removedfrom the explanted heart of a 46-year-old Caucasian male during a hearttransplantation. Patient history included myocardial infarction fromtotal occlusion of the left anterior descending coronary artery,atherosclerotic coronary artery disease, hyperlipidemia, myocardialischemia, dilated cardiomyopathy, and left ventricular dysfunction.Previous surgeries included cardiac catheterization. Family historyincluded atherosclerotic coronary artery disease. 199 3120415 LUNGTUT13The library was constructed using RNA isolated from tumorous lung tissueremoved from the right upper lobe of a 47-year-old Caucasian male duringa segmental lung resection. Pathology indicated invasive grade 3 (of 4)adenocarcinoma. Family history included atherosclerotic coronary arterydisease, and type II diabetes. 200 126758 LUNGNOT01 The library wasconstructed at Stratagene using RNA isolated from the lung tissue of a72-year-old male. 201 674760 CRBLNOT01 The library was constructed usingRNA isolated from the cerebellum tissue of a 69-year-old Caucasian malewho died from chronic obstructive pulmonary disease. Patient historyincluded myocardial infarction, hypertension, and osteoarthritis. 2021229438 BRAITUT01 The library was constructed using RNA isolated frombrain tumor tissue removed from a 50-year- old Caucasian female during afrontal lobectomy. Pathology indicated recurrent grade 3oligoastrocytoma with focal necrosis and extensive calcification.Patient history included a speech disturbance and epilepsy. Thepatient's brain had also been irradiated with a total dose of 5,082 cyg(Fraction 8). Family history included a brain tumor. 203 1236935LUNGFET03 The library was constructed using RNA isolated from lungtissue removed from a Caucasian female fetus who died at 20 weeks'gestation. 204 1359283 LUNGNOT12 The library was constructed using RNAisolated from lung tissue removed from a 78-year-old Caucasian maleduring a segmental lung resection and regional lymph node resection.Pathology indicated fibrosis pleura was puckered, but not invaded.Pathology for the associated tumor tissue indicated an invasivepulmonary grade 3 adenocarcinoma. Patient history includedcerebrovascular disease, arteriosclerotic coronary artery disease,thrombophlebitis, chronic obstructive pulmonary disease, and asthma.Family history included intracranial hematoma, cerebrovascular disease,arteriosclerotic coronary artery disease, and type I diabetes. 2051450703 PENITUT01 The library was constructed using RNA isolated fromtumor tissue removed from the penis of a 64- year-old Caucasian maleduring penile amputation. Pathology indicated a fungating invasive grade4 squamous cell carcinoma involving the inner wall of the foreskin andextending onto the glans penis. Patient history included benign neoplasmof the large bowel, atherosclerotic coronary artery disease, anginapectoris, gout, and obesity. Family history included malignantpharyngeal neoplasm, chronic lymphocytic leukemia, and chronic liverdisease. 206 1910668 CONNTUT01 The library was constructed using RNAisolated from a soft tissue tumor removed from the clival area of theskull of a 30-year-old Caucasian female. Pathology indicated chondroidchordoma with neoplastic cells reactive for keratin. 207 1955143CONNNOT01 The library was constructed using RNA isolated from mesenteryfat tissue obtained from a 71-year- old Caucasian male during a partialcolectomy and permanent colostomy. Family history includedatherosclerotic coronary artery disease, myocardial infarction, andextrinsic asthma. 208 1961637 BRSTNOT04 The library was constructedusing RNA isolated from breast tissue removed from a 62-year-old EastIndian female during a unilateral extended simple mastectomy. Pathologyfor the associated tumor tissue indicated an invasive grade 3 ductalcarcinoma. Patient history included benign hypertension, hyperlipidemia,and hematuria. Family history included cerebrovascular andcardiovascular disease, hyperlipidemia, and liver cancer. 209 1990762CORPNOT02 The library was constructed using RNA isolated from diseasedcorpus callosum tissue removed from the brain of a 74-year-old Caucasianmale who died from Alzheimer's disease. 210 1994131 CORPNOT02 Thelibrary was constructed using RNA isolated from diseased corpus callosumtissue removed from the brain of a 74-year-old Caucasian male who diedfrom Alzheimer's disease. 211 1997745 BRSTTUT03 The library wasconstructed using RNA isolated from breast tumor tissue removed from a58-year- old Caucasian female during a unilateral extended simplemastectomy. Pathology indicated multicentric invasive grade 4 lobularcarcinoma. The mass was identified in the upper outer quadrant, andthree separate nodules were found in the lower outer quadrant of theleft breast. Patient history included skin cancer, rheumatic heartdisease, osteoarthritis, and tuberculosis. Family history includedcerebrovascular disease, coronary artery aneurysm, breast cancer,prostate cancer, atherosclerotic coronary artery disease, and type Idiabetes. 212 2009035 TESTNOT03 The library was constructed using polyARNA isolated from testicular tissue removed from a 37- year-oldCaucasian male who died from liver disease. Patient history includedcirrhosis, jaundice, and liver failure. 213 2009152 TESTNOT03 Thelibrary was constructed using polyA RNA isolated from testicular tissueremoved from a 37- year-old Caucasian male who died from liver disease.Patient history included cirrhosis, jaundice, and liver failure. 2142061752 OVARNOT03 The library was constructed using RNA isolated fromovarian tissue removed from a 43-year-old Caucasian female duringremoval of the fallopian tubes and ovaries. Pathology for the associatedtumor tissue indicated grade 2 mucinous cystadenocarcinoma. Patienthistory included mitral valve disorder, pneumonia, and viral hepatitis.Family history included atherosclerotic coronary artery disease,pancreatic cancer, stress reaction, cerebrovascular disease, breastcancer, and uterine cancer. 215 2061933 OVARNOT03 The library wasconstructed using RNA isolated from ovarian tissue removed from a43-year-old Caucasian female during removal of the fallopian tubes andovaries. Pathology for the associated tumor tissue indicated grade 2mucinous cystadenocarcinoma. Patient history included mitral valvedisorder, pneumonia, and viral hepatitis. Family history includedatherosclerotic coronary artery disease, pancreatic cancer, stressreaction, cerebrovascular disease, breast cancer, and uterine cancer.216 2081422 UTRSNOT08 The library was constructed using RNA isolatedfrom uterine tissue removed from a 35-year-old Caucasian female during avaginal hysterectomy with dilation and curettage. Pathology indicatedthat the endometrium was secretory phase with a benign endometrial polyp1 cm in diameter. The cervix showed mild chronic cervicitis. Familyhistory included atherosclerotic coronary artery disease and type IIdiabetes. 217 2101278 BRAITUT02 The library was constructed using RNAisolated from brain tumor tissue removed from the frontal lobe of a58-year-old Caucasian male during excision of a cerebral meningeallesion. Pathology indicated a grade 2 metastatic hypernephroma. Patienthistory included a grade 2 renal cell carcinoma, insomnia, and chronicairway obstruction. Family history included a malignant neoplasm of thekidney. 218 2121353 BRSTNOT07 The library was constructed using RNAisolated from diseased breast tissue removed from a 43- year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology indicated mildly proliferative fibrocystic changes withepithelial hyperplasia, papillomatosis, and duct ectasia. Pathology forthe associated tumor tissue indicated invasive grade 4, nuclear grade 3mammary adenocarcinoma with extensive comedo necrosis. Family historyincluded epilepsy, cardiovascular disease, and type II diabetes. 2192241736 PANCTUT02 The library was constructed using RNA isolated frompancreatic tumor tissue removed from a 45- year-old Caucasian femaleduring radical pancreaticoduodenectomy. Pathology indicated a grade 4anaplastic carcinoma. Family history included benign hypertension,hyperlipidemia and atherosclerotic coronary artery disease. 220 2271935PROSNON01 This normalized prostate library was constructed from 4.4 Mindependent clones from the PROSNOT11 library. Starting RNA was madefrom prostate tissue removed from a 28-year-old Caucasian male who diedfrom a self-inflicted gunshot wound. The normalization and hybridizationconditions were adapted from Soares, M. B. et al. (1994) Proc. Natl.Acad. Sci. USA 91: 9228-9232, using a longer (19 hour) reannealinghybridization period. 221 2295344 BRSTNOT05 The library was constructedusing RNA isolated from breast tissue removed from a 58-year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology for the associated tumor tissue indicated multicentricinvasive grade 4 lobular carcinoma. Patient history included skincancer, rheumatic heart disease, osteoarthritis, and tuberculosis.Family history included cerebrovascular and cardiovascular disease,breast and prostate cancer, and type I diabetes. 222 2303994 BRSTNOT05The library was constructed using RNA isolated from breast tissueremoved from a 58-year-old Caucasian female during a unilateral extendedsimple mastectomy. Pathology for the associated tumor tissue indicatedmulticentric invasive grade 4 lobular carcinoma. Patient historyincluded skin cancer, rheumatic heart disease, osteoarthritis, andtuberculosis. Family history included cerebrovascular and cardiovasculardisease, breast and prostate cancer, and type I diabetes. 223 2497805ADRETUT05 The library was constructed RNA isolated from adrenal tumortissue removed from a 52-year-old Caucasian female during a unilateraladrenalectomy. Pathology indicated a pheochromocytoma. 224 2646362LUNGTUT11 The library was constructed using RNA isolated from lung tumortissue removed from the right lower lobe a 57-year-old Caucasian maleduring a segmental lung resection. Pathology indicated an infiltratinggrade 4 squamous cell carcinoma. Multiple intrapulmonary peribronchiallymph nodes showed metastatic squamous cell carcinoma. Patient historyincluded a benign brain neoplasm and tobacco abuse. Family historyincluded spinal cord cancer, type II diabetes, cerebrovascular disease,and malignant prostate neoplasm. 225 2657146 LUNGTUT09 The library wasconstructed using RNA isolated from lung tumor tissue removed from a68-year- old Caucasian male during segmental lung resection. Pathologyindicated invasive grade 3 squamous cell carcinoma and a metastatictumor. Patient history included type II diabetes, thyroid disorder,depressive disorder, hyperlipidemia, esophageal ulcer, and tobacco use.226 2755786 THP1AZS08 This subtracted THP-1 promonocyte cell linelibrary was constructed using 5.76 million clones from a5-aza-2′-deoxycytidine (AZ) treated THP-1 cell library. Starting RNA wasmade from THP- 1 promonocyte cells treated for three days with 0.8micromolar AZ. The hybridization probe for subtraction was derived froma similarly constructed library, made from RNA isolated from untreatedTHP-1 cells. 5.76 million clones from the AZ-treated THP-1 cell librarywere then subjected to two rounds of subtractive hybridization with 5million clones from the untreated THP-1 cell library. Subtractivehybridization conditions were based on the methodologies of Swaroop etal., NAR (1991) 19: 1954, and Bonaldo et al., Genome Research (1996) 6:791. THP-1 (ATCC TIB 202) is a human promonocyte line derived fromperipheral blood of a 1-year-old Caucasian male with acute monocyticleukemia. 227 2831245 TLYMNOT03 The library was constructed using RNAisolated from nonactivated Th1 cells. These cells were differentiatedfrom umbilical cord CD4 T cells with IL-12 and B7-transfected COS cells.228 3116250 LUNGTUT13 The library was constructed using RNA isolatedfrom tumorous lung tissue removed from the right upper lobe of a47-year-old Caucasian male during a segmental lung resection. Pathologyindicated invasive grade 3 (of 4) adenocarcinoma. Family historyincluded atherosclerotic coronary artery disease, and type II diabetes.229 3129630 LUNGTUT12 The library was constructed using RNA isolatedfrom tumorous lung tissue removed from a 70- year-old Caucasian femaleduring a lung lobectomy of the left upper lobe. Pathology indicatedgrade 3 (of 4) adenocarcinoma and vascular invasion. Patient historyincluded tobacco abuse, depressive disorder, anxiety state, and skincancer. Family history included cerebrovascular disease, congestiveheart failure, colon cancer, depressive disorder, and primary liver. 230007632 HMC1NOT01 The library was constructed using RNA isolated from theHMC-1 human mast cell line derived from a 52-year-old female. Patienthistory included mast cell leukemia. 231 1236968 LUNGFET03 The librarywas constructed using RNA isolated from lung tissue removed from aCaucasian female fetus who died at 20 weeks' gestation. 232 1334153COLNNOT13 The library was constructed using RNA isolated from ascendingcolon tissue of a 28-year-old Caucasian male with moderate chroniculcerative colitis. 233 1396975 BRAITUT08 The library was constructedusing RNA isolated from brain tumor tissue removed from the left frontallobe of a 47-year-old Caucasian male during excision of cerebralmeningeal tissue. Pathology indicated grade 4 fibrillary astrocytomawith focal tumoral radionecrosis. Patient history includedcerebrovascular disease, deficiency anemia, hyperlipidemia, epilepsy,and tobacco use. Family history included cerebrovascular disease andmalignant prostate neoplasm. 234 1501749 SINTBST01 The library wasconstructed using RNA isolated from ileum tissue removed from an18-year-old Caucasian female during bowel anastomosis. Pathologyindicated Crohn's disease of the ileum. Family history includedcerebrovascular disease and atherosclerotic coronary artery disease. 2351575240 LNODNOT03 The library was constructed using RNA isolated fromlymph node tissue removed from a 67-year- old Caucasian male during asegmental lung resection and bronchoscopy. This tissue was extensivelynecrotic with 10% viable tumor. Pathology for the associated tumortissue indicated invasive grade 3-4 squamous cell carcinoma. Patienthistory included hemangioma. Family history included atheroscleroticcoronary artery disease, benign hypertension, and congestive heartfailure. 236 1647884 PROSTUT09 The library was constructed using RNAisolated from prostate tumor tissue removed from a 66- year-oldCaucasian male during a radical prostatectomy, radical cystectomy, andurinary diversion. Pathology indicated grade 3 transitional cellcarcinoma. Patient history included lung neoplasm, and benignhypertension. Family history included malignant breast neoplasm,tuberculosis, cerebrovascular disease, atherosclerotic coronary arterydisease, and lung cancer. 237 1661144 BRSTNOT09 The library wasconstructed using RNA isolated from breast tissue removed from a45-year-old Caucasian female during unilateral extended simplemastectomy. Pathology for the associated tumor tissue indicated invasivenuclear grade 2-3 adenocarcinoma. Patient history included valvuloplastyof mitral valve and rheumatic heart disease. Family history includedcardiovascular disease and type II diabetes. 238 1685409 PROSNOT15 Thelibrary was constructed using RNA isolated from diseased prostate tissueremoved from a 66- year-old Caucasian male during radical prostatectomyand regional lymph node excision. Pathology indicated adenofibromatoushyperplasia. Pathology for the associated tumor tissue indicatedadenocarcinoma (Gleason grade 2 + 3). The patient presented withelevated prostate specific antigen (PSA). Family history includedprostate cancer, secondary bone cancer, and benign hypertension. 2391731419 BRSTTUT08 The library was constructed using RNA isolated frombreast tumor tissue removed from a 45-year- old Caucasian female duringunilateral extended simple mastectomy. Pathology indicated invasivenuclear grade 2-3 adenocarcinoma. Patient history included valvuloplastyof mitral valve and rheumatic heart disease. Family history includedcardiovascular disease and type II diabetes. 240 2650265 BRSTNOT14 Thelibrary was constructed using RNA isolated from breast tissue removedfrom a 62-year-old Caucasian female during a unilateral extended simplemastectomy. Pathology for the associated tumor tissue indicated aninvasive grade 3 (of 4), nuclear grade 3 (of 3) adenocarcinoma. Patienthistory included a benign colon neoplasm, hyperlipidemia, cardiacdysrhythmia, and obesity. Family history included cardiovascular andcerebrovascular disease and colon, ovary and lung cancer. 241 2677129KIDNNOT19 The library was constructed using RNA isolated from kidneytissue removed a 65-year-old Caucasian male during an exploratorylaparotomy and nephroureterectomy. Pathology for the associated tumortissue indicated grade 1 renal cell carcinoma within the upper pole ofthe left kidney. Patient history included malignant melanoma of theabdominal skin, benign neoplasm of colon, cerebrovascular disease, andumbilical hernia. Family history included myocardial infarction,atherosclerotic coronary artery disease, cerebrovascular disease, andprostate cancer. 242 3151073 ADRENON04 The normalized adrenal glandlibrary was constructed from 1.36 × 1e6 independent clones from anadrenal tissue library. Starting RNA was made from adrenal gland tissueremoved from a 20-year- old Caucasian male who died from head trauma.The library was normalized in two rounds using conditions adapted fromSoares et al. (PNAS (1994) 91: 9228-9232) and Bonaldo et al. (Genome Res(1996) 6: 791-806) using a significantly longer (48-hours/round)reannealing hybridization period. 243 3170095 BRSTNOT18 The library wasconstructed using RNA isolated from diseased breast tissue removed froma 57- year-old Caucasian female during a unilateral simple extendedmastectomy. Pathology indicated mildly proliferative breast disease.Patient history included breast cancer and osteoarthritis. Familyhistory included type II diabetes, gallbladder and breast cancer, andchronic lymphocytic leukemia. 244 3475168 LUNGNOT27 The library wasconstructed using RNA isolated from lung tissue removed from a17-year-old Hispanic female. 245 3836893 DENDTNT01 The library wasconstructed using RNA isolated from treated dendritic cells fromperipheral blood. 246 4072159 KIDNNOT26 The library was constructedusing RNA isolated from left kidney medulla and cortex tissue removedfrom a 53-year-old Caucasian female during a nephroureterectomy.Pathology for the associated tumor tissue indicated grade 2 renal cellcarcinoma involving the lower pole of the kidney. Patient historyincluded hyperlipidemia, cardiac dysrhythmia, menorrhagia,cerebrovascular disease, atherosclerotic coronary artery disease, andtobacco abuse. Family history included cerebrovascular disease andatherosclerotic coronary artery disease. 247 1003916 BRSTNOT03 Thelibrary was constructed using RNA isolated from diseased breast tissueremoved from a 54- year-old Caucasian female during a bilateral radicalmastectomy. Pathology for the associated tumor tissue indicated residualinvasive grade 3 mammary ductal adenocarcinoma. Patient history includedkidney infection and condyloma acuminatum. Family history includedbenign hypertension, hyperlipidemia and a malignant neoplasm of thecolon. 248 2093492 PANCNOT04 The library was constructed using RNAisolated from the pancreatic tissue of a 5-year-old Caucasian male whodied in a motor vehicle accident. 249 2108789 BRAITUT03 The library wasconstructed using RNA isolated from brain tumor tissue removed from theleft frontal lobe a 17-year-old Caucasian female during excision of acerebral meningeal lesion. Pathology indicated a grade 4 fibrillarygiant and small-cell astrocytoma. Family history included benignhypertension and cerebrovascular disease. 250 2171401 ENDCNOT03 Thelibrary was constructed using RNA isolated from dermal microvascularendothelial cells removed from a neonatal Caucasian male. 251 2212530SINTFET03 The library was constructed using RNA isolated from smallintestine tissue removed from a Caucasian female fetus, who died at 20weeks' gestation. 252 2253036 OVARTUT01 The library was constructedusing RNA isolated from ovarian tumor tissue removed from a 43- year-oldCaucasian female during removal of the fallopian tubes and ovaries.Pathology indicated grade 2 mucinous cystadenocarcinoma involving theentire left ovary. Patient history included mitral valve disorder,pneumonia, and viral hepatitis. Family history included atheroscleroticcoronary artery disease, pancreatic cancer, stress reaction,cerebrovascular disease, breast cancer, and uterine cancer. 253 2280161PROSNON01 The normalized prostate library was constructed from 4.4 Mindependent clones from the PROSNOT11 library. Starting RNA was madefrom prostate tissue removed from a 28-year-old Caucasian male who diedfrom a self-inflicted gunshot wound. The normalization and hybridizationconditions were adapted from Soares, M. B. et al. (1994) Proc. Natl.Acad. Sci. USA 91: 9228-9232, using a longer (19 hour) reannealinghybridization period. 254 2287485 BRAINON01 The library was constructedand normalized from 4.88 million independent clones from the BRAINOT03library. RNA was made from brain tissue removed from a 26-year-oldCaucasian male during cranioplasty and excision of a cerebral meningeallesion. Pathology for the associated tumor tissue indicated a grade 4oligoastrocytoma in the right fronto-parietal part of the brain. 2552380344 ISLTNOT01 The library was constructed using RNA isolated from apooled collection of pancreatic islet cells. 256 2383171 ISLTNOT01 Thelibrary was constructed using RNA isolated from a pooled collection ofpancreatic islet cells. 257 2396046 THP1AZT01 The library wasconstructed using RNA isolated from THP-1 promonocyte cells treated forthree days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB202)is a human promonocyte line derived from peripheral blood of a1-year-old Caucasian male with acute monocytic leukemia. 258 2456587ENDANOT01 The library was constructed using RNA isolated from aorticendothelial cell tissue from an explanted heart removed from a maleduring a heart transplant. 259 2484813 BONRTUT01 The library wasconstructed using RNA isolated from rib tumor tissue removed from a16-year-old Caucasian male during a rib osteotomy and a wedge resectionof the lung. Pathology indicated a metastatic grade 3 (of 4)osteosarcoma, forming a mass involving the chest wall. 260 2493851ADRETUT05 The library was constructed RNA isolated from adrenal tumortissue removed from a 52-year-old Caucasian female during a unilateraladrenalectomy. Pathology indicated a pheochromocytoma. 261 2495719ADRETUT05 The library was constructed RNA isolated from adrenal tumortissue removed from a 52-year-old Caucasian female during a unilateraladrenalectomy. Pathology indicated a pheochromocytoma. 262 2614153GBLANOT01 The library was constructed using RNA isolated from diseasedgallbladder tissue removed from a 53-year-old Caucasian female during acholecystectomy. Pathology indicated mild chronic cholecystitis andcholelithiasis with approximately 150 mixed gallstones. Family historyincluded benign hypertension. 263 2655184 THYMNOT04 The library wasconstructed using RNA isolated from thymus tissue removed from a3-year-old Caucasian male, who died from anoxia. Serologies werenegative. The patient was not taking any medications. 264 2848362BRSTTUT13 The library was constructed using RNA isolated from breasttumor tissue removed from the right breast of a 46-year-old Caucasianfemale during a unilateral extended simple mastectomy with breastreconstruction. Pathology indicated an invasive grade 3 adenocarcinoma,ductal type with apocrine features and greater than 50% intraductalcomponent. Patient history included breast cancer. 265 2849906 BRSTTUT13The library was constructed using RNA isolated from breast tumor tissueremoved from the right breast of a 46-year-old Caucasian female during aunilateral extended simple mastectomy with breast reconstruction.Pathology indicated an invasive grade 3 adenocarcinoma, ductal type withapocrine features and greater than 50% intraductal component. Patienthistory included breast cancer. 266 2899137 DRGCNOT01 The library wasconstructed using RNA isolated from dorsal root ganglion tissue removedfrom the cervical spine of a 32-year-old Caucasian male who died fromacute pulmonary edema and bronchopneumonia, bilateral pleural andpericardial effusions, and malignant lymphoma (natural killer celltype). Patient history included probable cytomegalovirus, infection,hepatic congestion and steatosis, splenomegaly, hemorrhagic cystitis,thyroid hemorrhage, and Bell's palsy. Surgeries included colonoscopy,large intestine biopsy, adenotonsillectomy, and nasopharyngeal endoscopyand biopsy; treatment included radiation therapy. 267 2986229 CARGDIT01The library was constructed using RNA isolated from diseased cartilagetissue. Patient history included osteoarthritis. 268 3222081 COLNNON03The normalized colon library was constructed from 2.84 × 10⁶ independentclones from the COLNNOT07 library. Starting RNA was made from colontissue removed from a 60-year-old Caucasian male during a lefthemicolectomy. The normalization and hybridization conditions wereadapted from Soares et al. (PNAS (1994) 91: 9228-9232), Swaroop et al.(Nucl. Acids Res. (1991) 19: 1954) and Bonaldo et al. (Genome Res (1996)6: 791-806), using a significantly longer (48 hour) reannealinghybridization period.

TABLE 5 Program Description Reference Parameter Threshold ABI FACTURA Aprogram that removes vector Perkin-Elmer Applied sequences and masksambiguous Biosystems, Foster City, CA. bases in nucleic acid sequences.ABI/PARACEL FDF A Fast Data Finder useful in Perkin-Elmer AppliedMismatch <50% comparing and annotating Biosystems, Foster City, CA;amino acid or nucleic acid Paracel Inc., Pasadena, CA. sequences. ABIAutoAssembler A program that assembles Perkin-Elmer Applied nucleic acidsequences. Biosystems, Foster City, CA. BLAST A Basic Local AlignmentAltschul, S. F. et al. (1990) ESTs: Probability value = Search Tooluseful in sequence J. Mol. Biol. 215: 403-410; 1.0E−8 or less similaritysearch for amino Altschul, S. F. et al. (1997) Full Length sequences:acid and nucleic acid sequences. Nucleic Acids Res. 25: Probabilityvalue = BLAST includes five functions: 3389-3402. 1.0E−10 or lessblastp, blastn, blastx, tblastn, and tblastx. FASTA A Pearson and Lipmanalgorithm Pearson, W. R. and D. J. ESTs: fasta E value = that searchesfor similarity Lipman (1988) Proc. Natl. 1.06E−6 between a querysequence and a Acad Sci. 85: 2444-2448; Assembled ESTs: fasta group ofsequences of the same Pearson, W. R. (1990) Methods Identity = 95% orgreater type. FASTA comprises as least Enzymol. 183: 63-98; and Matchlength = 200 five functions: fasta, tfasta, and Smith, T. F. and M. S.bases or greater; fastx, tfastx, and ssearch. Waterman (1981) Adv. Appl.fastx E value = 1.0E−8 Math. 2: 482-489. or less Full Length sequences:fastx score = 100 or greater BLIMPS A BLocks IMProved Searcher thatHenikoff, S and J. G. Henikoff, Score = 1000 or greater; matches asequence against those Nucl. Acid Res., 19: 6565-72, Ratio ofScore/Strength = in BLOCKS, PRINTS, DOMO, PRODOM, 1991. J. G. Henikoffand S. 0.75 or larger; and, if and PFAM databases to search for Henikoff(1996) Methods applicable, Probability gene families, sequence homology,Enzymol. 266: 88-105; and value = 1.0E−3 or less and structuralfingerprint regions. Attwood, T. K. et al. (1997) J. Chem. Inf. Comput.Sci. 37: 417-424. PFAM An algorithm for searching a query Krogh, A. etal. (1994) Score = 10-50 bits for sequence against hidden Markov modelJ. Mol. Biol., 235: 1501-1531; PFAM hits, depending on (HMM)-baseddatabases of protein Sonnhammer, E. L. L. et individual protein familiesfamily consensus sequences, such as PFAM. al. (1988) Nucleic Acids Res.26: 320-322. ProfileScan An algorithm that searches for Gribskov, M. etal. (1988) Score = 4.0 or greater structural and sequence motifs CABIOS4: 61-66; Gribskov, in protein sequences that match et al. (1989)Methods Enzymol. sequence patterns defined in 183: 146-159; Bairoch, A.Prosite. et al. (1997) Nucleic Acids Res. 25: 217-221. Phred Abase-calling algorithm that Ewing, B. et al. (1998) examines automatedsequencer Genome Res. 8: 175-185; traces with high sensitivity Ewing, B.and P. Green (1998) and probability. Genome Res. 8: 186-194. Phrap APhils Revised Assembly Program Smith, T. F. and M. S. Score = 120 orgreater; including SWAT and CrossMatch, Waterman (1981) Adv. Appl. Matchlength = 56 or programs based on efficient Math. 2: 482-489; Smith,greater implementation of the Smith- T. F. and M. S. Waterman Watermanalgorithm, useful in (1981) J. Mol. Biol. 147: searching sequencehomology 195-197; and Green, P., and assembling DNA sequences.University of Washington, Seattle, WA. Consed A graphical tool forviewing Gordon, D. et al. (1998) and editing Phrap assemblies GenomeRes. 8: 195-202. SPScan A weight matrix analysis program Nielson, H. etal. (1997) Score = 5 or greater that scans protein sequences for ProteinEngineering 10: 1-6; the presence of secretory signal Claverie, J. M.and S. Audic peptides. (1997) CABIOS 12: 431-439. Motifs A program thatsearches amino Bairoch et al. supra; acid sequences for patternsWisconsin Package Program that matched those defined in Manual, version9, page M51-59, Prosite. Genetics Computer Group, Madison, WI.

TABLE 6 Starting Ending Nucleotide Clone Fragment of NucleotideNucleotide SEQ ID NO: ID SEQ ID NO of Fragment of Fragment 135 443531443531H1 1 253 1406807F6 152 336 443531T6 847 355 SBBA00451F1 396 856SBBA00676F1 546 865 136 632860 632860H1 13 253 784715R3 17 666 509590H1455 706 137 670010 670010H1 1 263 669971R1 1 633 138 726498 726498H1 13263 726498R6 13 489 866599R3 7 660 139 795064 795064H1 86 323 4339458H14 284 937605R3 86 505 2381151F6 592 1057 1466346F6 857 1241 140 924925924925H1 111 412 3268330H1 2 239 759120R3 111 629 141 962390 1907958F6 1478 023569F1 1122 470 167282F1 1216 543 1309211F1 911 1224 142 12594051259405H1 46 277 2472425H1 331 354 774303R1 190 743 1520779F1 418 10011693833F6 914 1467 1831858T6.comp 1336 1742 1527737T6.comp 1386 1829 1431297384 1297384H1 402 641 1269310F6 1 492 1457367F1 792 1380 415587R11358 1712 SANA02967F1 1143 614 144 1299627 1299627H1 1 250 1359140F61004 1573 1349224F1 1330 1731 SBAA01431F1 46 397 SBAA02909F1 868 262SBAA01156F1 901 1266 145 1306026 1306026H1 1 223 1464088R6 302 829SBAA02496F1 92 568 SBAA04305F1 366 883 146 1316219 1316219H1 246 4912458603F6 1 402 2504756T6 980 380 147 1329031 1329031H1 1 264 1329031T6505 1 1329031F6 1 523 148 1483050 1483050H1 722 931 855049H1 1 267077017F1 1069 679 1483050F6 722 1215 1480024T6 2063 1315 1483050T6 20681535 759486R1 1762 2089 149 1514160 1514160H1 1640 1838 1866765T7 23832210 782676R1 1652 1875 008055X4 1090 1804 008055X5 1316 1952 1866765F62209 2391 SAOA03127F1 2129 1703 150 1603403 1603403H1 7 224 372910F1 42044 733299R7 219 420 151 1652303 1652303H1 4 256 1671806H1 1 2241341743T1 2069 1900 3803812H1 389 697 1878546F6 747 1344 1428640F1 10811664 2058609R6 1715 2098 1331621F1 1780 2096 1306331T1 1897 2098 1521693358 1693358H1 41 125 2498265H1 1 252 1867125F6 205 373 1693358T61094 416 2245848R6 737 1103 153 1707711 1707711H1 408 626 1484609T1 21651855 1707711F6 408 987 1267959F1 1721 2182 1484609F1 1855 2178SAJA00930F1 544 1132 SAJA01300R1 1675 1212 SAJA00999R1 1675 1142 1541738735 1738735H1 7 236 SAJA00944R1 393 5 SAJA00137F1 913 685SAJA03629F1 435 42 155 1749147 1749147H1 1 276 155 1749147F6 47 457 1551749147T6 479 1 156 1817722 1817722H1 1 268 2011085H1 344 545 1571831290 1831290H1 10 257 3473958H1 70 242 1972268F6 163 617 1301277F1413 852 1521574F1 1024 1602 1561690T6 1729 1058 891461R1 1261 1738 1581831477 1831477H1 59 337 1582867H1 1 199 1336769T1 1986 1639 1933092H1525 789 1519909F1 841 1296 1220946H1 1061 1318 809556T1 1983 16871217559T1 2002 1445 1309225F1 1747 2001 159 1841607 1841607H1 13 192SBHA03588F1 13 172 160 1852391 1852391H1 98 367 734140H1 1 225 1852391F698 542 161 1854555 1854555H1 1 265 2511711H1 37 58 782453R1 223 7121854555F6 1 346 1840675T6 1046 860 2109736H1 938 1054 162 18557551855755H1 17 224 3040236H1 1 179 1283207F1 306 816 833763T1 1148 8351920926R6 854 1161 163 1861434 1861434H1 13 253 1861434T6 872 261SARA01525F1 426 808 SARA02548F1 587 889 164 1872334 1872334H1 1 2291872334F6 1 424 SBGA03684F1 358 425 165 1877230 1877230H1 1405 16772519841H1 1 251 1877230T6 1903 1405 1254693F1 335 716 077020R1 682 14141232336F1 906 1507 1004952R6 1451 1904 SARA01879F1 1545 1921 SARA02654F11545 1923 166 1877885 1877885H1 68 323 508020F1 499 51 2751126R6 219 516SARA02571F1 407 499 167 1889269 1889269H1 757 1020 1915551H1 1 191629493X12 481 865 1441289F1 693 865 1215274X34F1 1106 1631 1818447F61307 1540 1208463R1 1372 1493 168 1890243 1890243H1 9 268 SARA01884F1521 168 SATA00046F1 1057 851 SARA03294F1 1329 910 SARA02790F1 1138 1535169 1900433 1900433H1 1 242 SATA00396F1 409 124 SATA02742F1 1 294 1701909441 1909441H1 786 1048 1398811F1 1 550 3039939H1 607 876 3324740H1685 944 1442131F6 787 1232 2254056H1 1423 1522 2199453T6 1955 13511698531H1 1968 1796 171 1932226 1932226H1 294 510 2320569H1 1 2661932226F6 294 685 2469455T6 1475 1071 2469455F6 1034 1492 1907140F6 11581482 SATA02592F1 857 518 172 1932647 1932647H1 17 246 1492745T1 15821418 1492745H1 1418 1599 SASA02355F1 386 19 SASA00117F1 250 569SASA00192F1 515 816 173 2124245 2124245H1 45 190 1235393F1 495 8951402264F6 323 925 1303990F1 682 1240 1402264T6 1613 950 174 21326262132626H1 406 651 1723432T6 1299 746 2132626R6 406 904 1736723T6 1292857 1504738F1 868 1320 175 2280639 2280639H1 28 303 1377560F6 261 777176 2292356 2292356H1 717 968 4086827H1 1 275 1754442F6 232 5773571126H1 497 808 1601305F6 808 1464 177 2349310 2349310H1 1 2362349310T6 682 2 178 2373227 2373227H1 298 524 3316444H1 801 1053302685R6 1141 1496 SASA02181F1 577 1 SASA01923F1 963 466 SASA03516F11102 1249 179 2457682 2457682H1 1 226 2457682F6 1 554 180 24804262480426H1 1 213 2480426F6 1 501 181 2503743 2503743H1 6 222 1853909H1 1272 1517619F1 172 830 1467896F6 540 1112 490031F1 1647 1068 1208654R11382 1633 880544R1 1450 1648 182 2537684 2537684H1 434 682 2005493H1 1194 730969H1 307 547 916487H1 723 989 996135R1 997 1598 1920738R6 13061692 1957710F6 1472 1692 183 2593853 2593853H1 1 252 807497H1 2 217914020R6 284 740 889992R1 416 729 184 2622354 2622354H1 3 266 2623992H11 246 1556510F6 81 258 185 2641377 2641377H1 126 369 4341415H2 10 345SBCA07049F3 126 599 186 2674857 2674857H1 139 393 1872373H1 1 270470512R6 1486 1502 1728547H1 1285 1508 3013651F6 1423 1987 SBCA01366F1819 385 SBCA00694F1 973 1198 187 2758485 2758485H1 20 267 3097533H1 1158 1578959F6 291 771 188 2763296 2763296H1 63 301 3486025F6 1 130SBDA07002F3 63 687 189 2779436 2779436H1 1 233 2779436F6 1 577SBDA07009F3 1 608 190 2808528 2808528H1 25 335 2611513F6 2 489SBDA07021T3 1058 443 191 2809230 2809230H1 409 630 2213849H1 1 133711706R6 396 691 958323R1 407 800 030732F1 1366 623 192 28168212816821H1 210 501 3746964H1 1 307 2816821F6 210 682 948722T6 959 527 1932817268 2817268H1 42 282 3591308H1 13 264 419522R1 179 808 2073028F6 446924 1308781F6 869 1112 194 2923165 2923165H1 8 295 2011630H1 18 2381457250F1 268 856 754668R1 327 878 1406510F6 558 901 195 29498222949822H1 1 280 SBDA07078F3 1 606 196 2992192 2992192H1 25 321 2534324H21 240 2815255T6 690 219 1551107T6 893 471 1551107R6 471 690 197 29924582992458H1 48 362 2618951H1 1 247 1479252F1 163 610 1879054H1 563 8401879054F6 563 1096 2215240H1 951 1202 1535968T1 1729 1173 198 30447103044710H1 652 952 3741773H1 1 283 859906X42C1 94 192 1534347F1 90 2681421122F1 830 1392 1303865F1 1033 1487 1704452F6 1432 1934 1251642F12006 1544 1781694R6 1894 2017 199 3120415 3120415H1 72 363 1360123T1 523141 1375015H1 380 526

1. An isolated polynucleotide encoding a polypeptide comprising an aminoacid sequence having at least about 95% sequence identity to an aminoacid sequence of SEQ ID NO: 43, wherein the homologous polypeptide has asilent change in its amino acid sequence relative to the polypeptiderepresented by SEQ ID NO:
 43. 2. The isolated polynucleotide of claim 1,wherein the polynucleotide encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 43. 3. The isolated polynucleotide of claim1, comprising a polynucleotide sequence of SEQ ID NO:
 177. 4. Arecombinant polynucleotide comprising a promoter sequence operablylinked to the polynucleotide of claim
 1. 5. An isolated cell transformedwith the recombinant polynucleotide of claim
 4. 6. A method of producinga polypeptide comprising: a) culturing a cell under conditions suitablefor expression of the polypeptide, wherein the cell is transformed witha recombinant polynucleotide, and the recombinant polynucleotidecomprises a promoter sequence operably linked to the polynucleotide ofclaim 1, and b) recovering the polypeptide so expressed.
 7. The methodof claim 6, wherein the recombinant polynucleotide comprises thepolynucleotide sequence of SEQ ID NO:
 177. 8. An isolated polynucleotidecomprising a polynucleotide sequence selected from the group consistingof: a) a polynucleotide sequence having at least about 95% sequenceidentity to a polynucleotide sequence of SEQ ID NO: 177, wherein thehomologous polynucleotide encodes a polypeptide with at least onefunctional characteristic of the polypeptide encoded by thepolynucleotide sequence of SEQ ID NO: 177; b) a polynucleotide sequencecomplementary to the polynucleotide sequence of a); and c) an RNAequivalent of the polynucleotide sequence of a) or b).
 9. The isolatedpolynucleotide of claim 8, comprising the polynucleotide sequence of SEQID NO:
 177. 10. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO: 43; b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical to the amino acid sequence of SEQID NO: 43; c) a biologically active fragment of a polypeptide having theamino acid sequence of SEQ ID NO: 43; and d) an immunogenic fragment ofa polypeptide having the amino acid sequence of SEQ ID NO:
 43. 11. Theisolated polypeptide of claim 10, comprising a naturally occurring aminoacid sequence at least 95% identical to the amino acid sequence of SEQID NO:
 43. 12. The isolated polypeptide of claim 10, comprising an aminoacid sequence of SEQ ID NO: 43.